Cell-free sensor systems

ABSTRACT

The present described inventions relate, inter alia, to methods and compositions that provide for improved detection of target molecules in, for example, bioengineering.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos.62/375,305, filed Aug. 15, 2016; 62/375,301, filed Aug. 15, 2016;62/378,999, filed Aug. 24, 2016; and 62/379, 002, filed August 24, 2016,the contents of which are hereby incorporated by reference herein intheir entirety.

FIELD

The present described inventions relate, inter alia, to methods andcompositions that provide for a cell-free system for engineering anddeploying allosteric sensor proteins.

BACKGROUND

A key objective of synthetic biology is the efficient production of highvalue target molecules. But, a significant unsolved bottleneck in thebioengineering design-build-test cycle is in the test phase due toscreening limitations. One possible solution to this bottleneck is theuse of molecular sensors. Indeed, sensors that recognize industriallyimportant molecules are rapidly becoming part of metabolic engineeringstrategies to improve enzymatic bioproduction and detection. However,coupling a response to the detection of a specific target is anengineering challenge in itself.

The use of allosteric proteins—single proteins that directly couple therecognition of a molecule of interest to a response has been proposed.Allostery is a common feature of proteins, in which the behavior at an‘active’ site is altered by binding of an effector to a second or‘allosteric’ site, often quite distant from the first (about 10A ormore). The altered behavior can either directly or indirectly lead to achange in the protein's activity and thereby elicit a detectableresponse.

The use of bacterial allosteric transcription factors (aTFs)—singleproteins that directly couple the recognition of a small molecule to atranscriptional output—has been proposed (Taylor, et al. Nat. Methods13(2): 177). The protein's conformational change caused by effectorbinding modulates its affinity for a specific operator DNA sequence,which alters gene expression by up to 5000-fold. Any strategy toengineer aTF sensors for new molecular recognition engineers both thesensing and actuation functions that are needed for a sensing device tooperate. This makes aTF sensors an exciting paradigm to address thesense-and-respond challenge that is central to many applications ofsynthetic biology.

The use of circulary permuted reporter proteins about the active site ofa second conformationally dynamic effector protein presents analternative method for directly coupling the recognition of a moleculeto a response.

The protein's conformational change caused by effector binding resultsin a shift in protein structure that can be repurposed to switch areporter protein from an inactive to active state or vice versa. Thisresponse can either be positive or negative as well as stoichiometric oramplifiable. For example, the circular permutation of GFP about thebinding site of a protein has resulted in a fluorescent state that isdirectly coupled to the binding of a small molecule effector. Since oneprotein may bind a specific number of molecules based on its structureand relative affinity, the result leads to a stoichiometric fluorescentsignal directly correlated to the amount of ligand-bound proteinpresent. Permutation of an enzyme about the active site on the otherhand results in signal amplification as a single effector molecule leadsto multiple functional turnovers for the reporter enzyme. Further,effector modulated presentation of a degradation tag results in theselective reduction in a protein that may either have a beneficial orderogatory effect on cellular state.

One of the challenges of engineering sensor proteins—such as aTFs,circularly permuted reporter-binders, and allosterically controlleddegradation tags—to recognize target molecules is that the host cell inwhich the molecular biology is conducted may not permit sufficientlyadjustable concentrations of the target molecule to allow a measurableon/off response of the engineered protein. Simple introduction of thetarget molecule into the growth medium exogenously or throughbioproduction may not be a suitable approach because the target moleculemay be excluded from, transported out of, toxic to, or chemicallyaltered by the cell or the target molecule's concentration is activelycontrolled by the cell. These active or passive mechanisms modulatingthe effective concentration of the ligand convolutes the sensor'sability to respond to the true concentration of the ligand being addedor produced.

Furthermore, proteins are often sensitive to deviations in theirenvironmental conditions—such as buffer compositions, metaboliteprofiles, temperature, etc.—that may lead to deviations in proteinactivity. Allosteric proteins also suffer to various degrees to thisphenomenon. As a result, their phenotypic sensitivity to cellularenvironment when used as a biological sensor system has the potential toskew results as the environmental conditions are artificially orbiologically adjusted.

Further, protein engineering usually occurs through discrete steps infunction from wild type to desired activity. In the case of ligandbinding, both affinity and specificity for a target molecule is usuallygained in incremental steps that tend to weaken affinity and broadenspecificity before the wild-type activity is lost. This phenomenonpresents a specific challenge for engineering biosensors within cellswhere the natural ligands will likely be present and therefore generatea detectable sensor response that may convolute the desired response.This interference may obstruct identification of sensors with low tomedium activity for the desired ligand as the response to the wild typeligand may be greater than the response to the targeted ligand. Thisinterference may also present itself when deploying engineered sensorsas the wild type ligand response may obstruct the response to thedesired metabolite. Therefore, depending on the ubiquity of the nativeligand, there is a need to separate the sensor system from the wild-typeenvironment in the beginning steps of the sensor engineering process aswell as in their deployment.

This same challenge presents itself when engineering a sensor's DNAbinding sites to recognize a new DNA sequence for allosterictranscription factors and their response regulators formultiple-component systems. In order to substantially change the DNAbinding site (operator site) specificity to a completely novel andforeign site often requires iterative steps that transition inspecificity from wild-type, to broad specificity, to new target.

Additionally, these intermediate steps may demonstrate off-targeteffects by binding to unknown and unpredictable locations leading tounwarranted changes in cellular state. Therefore, intermediate steps forengineering operator sites may be required to be performed in anacellular environment.

As a result, there is a need for improved compositions and methods forboth developing engineered allosteric sensor proteins as well asdeploying them for sensing target molecules in a manner not limited bythe cellular environment.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic of the methods/systems of non-limitingembodiments of the invention. Specifically, panel A shows a strategy fortemporal control in the reporter assays using a orthogonal sensor forengineered tetR sensors in the case where the engineered sensor isnon-functional and panel B shows a strategy for temporal control in thereporter assays using a orthogonal sensor for engineered tetR sensors inthe case where the engineered sensor is functional.

FIG. 2 shows an illustrative overview of the cell-free transcriptionfactor screening strategy using bulk emulsions. Specifically, panel Ashows a pool of allosteric transcription factors (aTF) expressed in E.coli and encapsulated in water-in-oil droplets generated in bulk. Eachdroplet contains the effector of interest, the polymerase reporter DNAunder negative control through the aTF operator, primers specific to theaTF gene, and a chemical or enzymatic lytic agent. Panel B shows a moredetailed view of the reporter strategy. aTFs that respond to theeffector result in production of Kod polymerase that is then utilized toamplify the aTF genotype by PCR. Enriched amplicons are recovered fromthe emulsion and then cloned back into their expression vectors forsubsequent rounds of screening.

FIG. 3 shows an overview of the cell-free transcription factor screeningstrategy using microfluidically generated emulsions. Specifically, panelA shows a pool of allosteric transcription factors (aTF) are expressedin E. coli and encapsulated in water in oil droplets generatedmicrofluidically. The effector, polymerase reporter gene, primers, andlytic agent are introduced through a second internal aqueous inlet. aTFsare enriched using the strategy presented in Figure (panel b), panel Band C show photographs of the droplet production chips from DolomiteMicrofluidics for reference, but any microfluidic chip may be used.Panel D shows schematic of the flow focusing junction producingwater-in-oil droplets. Panel E shows photograph of water-in-oil dropletformation. The channel width is 14 μm for scale, and panel F showswater-in-oil droplets are stable for more than 1 week at 37° C. and aremonodisperse with a size of ˜15±1 μm.

FIG. 4 shows an illustrative overview of the cell-free RNAtranscription-based reporter strategy. A pool of allosterictranscription factors (aTF) are expressed in E. coli and encapsulated inwater in oil droplets either microfluidically or in bulk. The effector,IVT reagents, and lytic agent re-introduced separately. Afterencapsulation, the droplets are incubated at 37° C. to promote lysisreleasing the aTFs. aTFs that respond to the effector result in RNAtranscription that is then utilized to amplify the aTF genotype byRT-PCR. Enriched amplicons are recovered from the emulsion and thencloned back into their expression vectors for subsequent rounds ofscreening. This strategy may replace the DNA polymerase strategy in FIG.2 and FIG. 3.

FIG. 5 shows an overview of the cell-free transcription factor screeningstrategy using microfluidically generated double emulsions.Specifically, panel A shows a pool of allosteric transcription factors(aTF) are expressed in E. coli and encapsulated in water in oil dropletsgenerated in bulk. Each droplet contains the effector of interest, theenzyme reporter DNA under negative control through the aTF operator, afluorogenic substrate, and a chemical or enzymatic lytic agent. Afterencapsulation, the droplets are incubated at 37° C. to promote lysisreleasing the aTFs. aTFs that respond to the effector result inproduction of the reporter enzyme. The single emulsions are thenconverted into a water-in-oil double emulsion and sorted by FACS, panelB shows the water-in-oil emulsions either receive no E. coli, receive anE. coli expressing an unresponsive aTF, or an E. coli containing aresponsive aTF resulting in the production of fluorescent signal, panelC shows photograph of double emulsion formation. The channel width is 14pm for scale, panel D shows photograph of water-in-oil-in-water dropletsthat are stable for more than 1 week at 24° C. and are monodisperse witha size of ˜20±2.2 μm, and panel E shows Schematic of the secondemulsion. d, Photograph of double emulsion formation.

FIG. 6 shows single chip water-in-oil-in-water formation for cell-freetranscription factor screening. Specifically, panel A shows a schematicrepresentation of the chip design, and panel B shows a photograph of thesingle chip design in PDMS producing double emulsions in one step.channel width is 50 μm for scale.

FIG. 7 shows sensitivity and dynamic range of beta-glucosidasefluorogenic reporter substrate.

FIG. 8 shows sensitivity and dynamic range of Antarctic phosphatase (AP)fluorogenic reporter substrate.

FIG. 9 shows aTF-dependent control of T7 transcription in vitro.

FIG. 10 shows the dose response of 4 TetR sensors engineered to detectthe target molecule nootkatone (CE3, GF1, GA3, and CG5) and wild typeTetR (p523) to nootkatone and ATc.

FIG. 11 shows flow cytometry data of p1174 plasmid causing loss of thep1057 target plasmid

FIG. 12 shows dilutions of cultures on selective media for either p1174or p1057 to estimate loss of carb plasmid.

SUMMARY

Accordingly, in general, methods and compositions that improve thedevelopment of engineered, allosteric sensor proteins, such asengineered aTFs, as wells their utility in detection and/or productionof target molecules in cell-free environments are provided. Furthermore,engineered sensors are not limited to their utility within theenvironment in which they were derived, i.e. cellularly derived sensorsmay also be deployed in acellular environments and vice versa.Accordingly, the present invention provides compositions and methodsthat allow for the detection and/or production of target molecules andcan be produced in manners that are independent of limiting processes ofa cell and therefore not contingent on, for example, retention of thetarget molecule within a cell, e.g. a healthy cell.

In various embodiments, the present invention is not necessarily limitedby an inherent toxicity of the target molecules to a cell, the abilityof any target molecule to enter or remain inside screening strain cells,or the ability of any target molecule to be unaltered by cellularmachinery. Further, the present invention is not limited by the sensingof molecules either small or large, but may be extended to cellularstates such as redox potential and charge. Further, the presentinvention is not limited to the utility of allosteric transcriptionfactors that directly bind to a DNA operator, but may use effectordomains that propagate though protein cascades such as two componentsystems. Accordingly, the present methods and compositions allow formeasurable on/off response of the engineered protein that is not limitedby the ability of a cell to withstand or maintain measurableconcentrations.

In one aspect, the present invention relates to compositions and methodsfor making an engineered protein sensor and/or switch, e.g. from anallosteric protein, e.g. a transcription factor, that binds to andallows detection of a target molecule, wherein the engineered proteinsensor and/or switch is produced and screened at least in part,acellularly, and/or allows target molecules to be screened eithercellularly or acellularly.

In one embodiment, there is a provided method of making an allostericsensor and/or switch that binds controllably to a ligand different fromthat of the wild type ligand. The engineered sensor and/or switch bindsto and allows detection of the target molecule through a detectableresponse wherein the engineered protein sensor and/or switch is producedand screened at least in part, acellularly, and/or allows targetmolecules to be screened acellularly not limited to methods as describedabove.

In a further embodiment, there is a provided method of making anallosteric sensor and/or switch that binds controllably to an engineeredDNA sequence different from that of the wild type sequence in responseto the binding of a target molecule. The engineered sensor and/or switchbinds to and allows detection of a target molecule through binding to anengineered DNA sequence wherein the engineered protein sensor and/orswitch is produced and screened at least in part, acellularly, and/orallows target molecules to be screened acellularly not limited tomethods as described above.

In various embodiments, the allosteric sensor and or switch may beengineered to recognize both a new ligand as well as a new DNA bindingsite simultaneously.

In another aspect, the present invention relates to compositions andmethods for deploying sensors and/or switches to detect the productionof target molecules. In various embodiments, the engineered sensorand/or switch developed acellularly may be used either in a cellular oracellular environment. In further embodiments, an engineered sensorand/or switched developed cellularly may be used in an acellularenvironment.

In various aspects, the present invention relates to a method of makingan allosteric DNA-binding protein sensor and/or switch which binds to atarget molecule. The method comprises steps of (a) constructing acandidate allosteric DNA-binding protein sensor and/or switch, theconstructing comprising (i) designing a DNA-binding protein sensorand/or switch for an ability to bind a target molecule, the designingoptionally being in silico or (ii) undertaking directed or randommutagenesis to yield a candidate allosteric DNA-binding protein sensorand/or switch having an ability to bind a target molecule; (b) providinga host cell with a nucleic acid encoding the candidate allostericDNA-binding protein sensor and/or switch and a nucleic acid encoding areporter gene system and selecting for a cell comprising the candidateallosteric DNA-binding protein sensor and/or switch and the reportergene system; (c) isolating nucleic acids from the cell comprising thecandidate allosteric DNA-binding protein sensor and/or switch and thereporter gene system and contacting the isolated nucleic acids with anin vitro transcription (IVT) or an in vitro transcription andtranslation (IVTT) mixture, the IVT or IVTT mixture comprising a targetmolecule and a detection reagent; and (d) interrogating the IVT or IVTTmixture for reporter response, the reporter response being indicative oftarget molecule binding to the candidate allosteric DNA-binding proteinsensor and/or switch.

In various embodiments, the allosteric DNA-binding protein sensor and/orswitch is an engineered prokaryotic transcriptional regulator familymember optionally selected from a LysR, AraC/XylS, TetR, LuxR, Lacl,ArsR, MerR, AsnC, MarR, NtrC (EBP), OmpR, DeoR, Cold shock, GntR, andCrp family member.

In various embodiments, the target molecule is a small molecule that isnot a native ligand of the wild type candidate allosteric DNA-bindingprotein sensor and/or switch.

In various embodiments, the target molecule is an antibiotic.

In various embodiments, step (a) comprises mutating an allostericprotein.

In various embodiments, the nucleic acid is provided to the host cell byone or more of electroporation, chemical transformation, ballistictransformation, pressure induced transformation, electrospray injection,mechanical shear forces induced, for example, in microfluids, and carbonnanotubes, nanotube puncture, induced natural competence mechanisms ofan organism, merging of protoplasts, and conjugation with Agrobacterium.

In various embodiments, the host cell is selected from a eukaryotic orprokaryotic cell, selected from a bacterial, yeast, algal, plant,insect, mammalian cells, and immortalized cell.

In various embodiments, the reporter gene system comprises a proteinhaving a unique spectral signature and/or assayable enzymatic activity.

In various embodiments, the IVT or IVTT mixture comprises a coupled orlinked system.

In various embodiments, the reporterresponse is a direct amplificationof the genotype of the allosteric protein.

In various embodiments, the nucleic acid encoding the candidateallosteric DNA-binding protein sensor and/or switch and the nucleic acidencoding the reporter gene system comprises a single nucleic acidvector.

In various embodiments, the nucleic acid encoding the candidateallosteric DNA-binding protein sensor and/or switch and the nucleic acidencoding the reporter gene system comprises two nucleic acid vectors.

In various embodiments, the method further comprises a step of (e):isolating the nucleic acid encoding the candidate allosteric DNA-bindingprotein sensor and/or switch, e.g., comprising use of flasks, culturetubes, and plastic ware, microliter plates, patterned microwells, ormicrodroplets generated either in bulk or microfluidically.

In various aspects, the present invention relates to a method of makingan allosteric DNA-binding protein sensor and/or switch which binds to atarget molecule. The method comprises steps of (a) constructing acandidate allosteric DNA-binding protein sensor and/or switch, theconstructing comprising (i) designing a DNA-binding protein sensorand/or switch for an ability to bind a target molecule, the designingoptionally being in silico or (ii) undertaking directed or randommutagenesis to yield a DNA-binding protein sensor and/or switch whichhas an ability to bind a target molecule; (b) providing a host cell witha nucleic acid encoding the candidate allosteric DNA-binding proteinsensor and/or switch and a nucleic acid encoding a reporter gene systemand selecting for a cell comprising the candidate allosteric DNA-bindingprotein sensor and/or switch and the reporter gene system; (c) isolatingnucleic acids from the cell comprising the candidate allostericDNA-binding protein sensor and/or switch and the reporter gene systemand contacting the isolated nucleic acids with an in vitro transcription(IVT) or an in vitro transcription and translation (IVTT) mixture, theIVT or IVTT mixture comprising a target molecule and a detectionreagent; and (d) interrogating the IVT or IVTT mixture by nucleic acidsequencing before and after selection to determine those molecules thathave become functionally enriched.

In various embodiments, the allosteric DNA-binding protein sensor and/orswitch is an engineered prokaryotic transcriptional regulator familymember optionally selected from a LysR, AraC/XylS, TetR, LuxR, Lacl,ArsR, MerR, AsnC, MarR, NtrC (EBP), OmpR, DeoR, Cold shock, GntR, andCrp family member.

In various embodiments, the target molecule is a small molecule that isnot a native ligand of the wild type candidate allosteric DNA-bindingprotein sensor and/or switch.

In various embodiments, the target molecule is an antibiotic.

In various embodiments, step (a) comprises mutating an allostericprotein.

In various embodiments, the nucleic acid is provided to the host cell byone or more of electroporation, chemical transformation, ballistictransformation, pressure induced transformation, electrospray injection,mechanical shear forces induced, for example, in microfluids, and carbonnanotubes, nanotube puncture, induced natural competence mechanisms ofan organism, merging of protoplasts, and conjugation with Agrobacterium.

In various embodiments, the host cell is selected from a eukaryotic orprokaryotic cell, selected from a bacterial, yeast, algal, plant,insect, mammalian cells, and immortalized cell.

In various embodiments, the reporter gene system comprises a proteinhaving a unique spectral signature and/or assayable enzymatic activity.

In various embodiments, the IVT or IVTT mixture comprises a coupled orlinked system.

In various embodiments, the reporter response is a direct amplificationof the genotype of the allosteric protein.

In various embodiments, the nucleic acid encoding the candidateallosteric DNA-binding protein sensor and/or switch and the nucleic acidencoding the reporter gene system comprises a single nucleic acidvector.

In various embodiments, the nucleic acid encoding the candidateallosteric DNA-binding protein sensor and/or switch and the nucleic acidencoding the reporter gene system comprises two nucleic acid vectors.

In various embodiments, the method further comprises a step of (e):isolating the nucleic acid encoding the candidate allosteric DNA-bindingprotein sensor and/or switch, e.g., comprising use of flasks, culturetubes, and plastic ware, microliter plates, patterned microwells, ormicrodroplets generated either in bulk or microfluidically.

In various aspects, the present invention relates to a method of makingan allosteric DNA-binding protein sensor and/or switch which binds to atarget molecule. The method comprising steps of (a) constructing acandidate allosteric DNA-binding protein sensor and/or switch, theconstructing comprising (i) designing a DNA-binding protein sensorand/or switch for an ability to bind a target molecule, the designingoptionally being in silico or (ii) undertaking directed or randommutagenesis to yield the candidate allosteric DNA-binding protein sensorand/or switch having an ability to bind a target molecule; (b)contacting a solid support with a nucleic acid encoding the candidateallosteric DNA-binding protein sensor and/or switch and selecting for asolid support comprising the candidate allosteric DNA-binding proteinsensor and/or switch; (c) isolating nucleic acids from the solid supportcomprising the candidate allosteric DNA-binding protein sensor and/orswitch and contacting the isolated nucleic acids with an in vitrotranscription (IVT) or an in vitro transcription and translation (IVTT)mixture; (d) introducing a reporter gene system, detection reagent, andtarget molecule, and interrogating the mixture for a reporter response,the reporter response being indicative of the target molecule binding tothe candidate allosteric DNA-binding protein sensor and/or switch.

In various embodiments, the solid support is a nanoparticle and amicroparticle, a bead, a nanobead, a microbead, or an array.

In various embodiments, the candidate allosteric DNA-binding proteinsensor and/or switch is an engineered prokaryotic transcriptionalregulator family member optionally selected from a LysR, AraC/XylS,TetR, LuxR, Lacl, ArsR, MerR, AsnC, MarR, NtrC (EBP), OmpR, DeoR, Coldshock, GntR, and Crp family member.

In various embodiments, the target molecule is a small molecule that isnot a native ligand of the wild type candidate allosteric DNA-bindingprotein sensor and/or switch.

In various embodiments, the target molecule is an antibiotic.

In various embodiments, step (a) comprises mutating an allostericprotein.

In various embodiments, the reporter gene system comprises a proteinhaving a unique spectral signature and/or assayable enzymatic activity.

In various embodiments, the IVT or IVTT mixture comprises a coupled orlinked system.

In various embodiments, the reporter response is a direct amplificationof the genotype of the allosteric protein.

In various embodiments, the nucleic acid encoding the candidateallosteric DNA-binding protein sensor and/or switch and the nucleic acidencoding the reporter gene system comprises a single nucleic acidvector.

In various embodiments, the nucleic acid encoding the candidateallosteric DNA-binding protein sensor and/or switch and the nucleic acidencoding the reporter gene system comprises two nucleic acid vectors.

In various embodiments, the nucleic acid encoding the candidateallosteric DNA-binding protein sensor and/or switch comprises asynthetic DNA, amplified DNA, or amplified RNA.

In various embodiments, the method further comprises a step of (e):isolating the nucleic acid encoding the allosteric DNA-binding proteinsensor and/or switch, e.g., comprising use of flasks, culture tubes, andplastic ware, microliter plates, patterned microwells, or microdropletsgenerated either in bulk or microfluidically.

In various aspects, the present invention relates to a method for makinga target molecule in a biological cell. The method comprises steps of(a) engineering the biological cell to produce the target molecule; (b)introducing an allosteric DNA-binding protein sensor and/or switch whichbinds to the target molecule in the biological cell;

and (c) screening for target molecule production.

In embodiments, the biological cell is engineered to produce the targetmolecule by a multiplex genome engineering technique and/or a methodinvolving a double-strand break (DSB) or single-strand break or nick.

In various embodiments, the allosteric DNA-binding protein sensor and/orswitch which binds to the target molecule is produced by a methodcomprising steps of (a) constructing a candidate allosteric DNA-bindingprotein sensor and/or switch, the constructing comprising (i) designinga candidate allosteric DNA-binding protein sensor and/or switch for anability to bind the target molecule, the designing optionally being insilico or (ii) undertaking directed or random mutagenesis to yield thecandidate allosteric DNA-binding protein sensor and/or switch having anability to bind the target molecule; (b) providing a host cell with anucleic acid encoding the candidate allosteric DNA-binding proteinsensor and/or switch and a nucleic acid encoding the reporter genesystem and selecting for a cell comprising the candidate allostericDNA-binding protein sensor and/or switch and the reporter gene system;(c) isolating nucleic acids from the cell comprising the candidateallosteric DNA-binding protein sensor and/or switch and the reportergene system and contacting the isolated nucleic acids with an in vitrotranscription (IVT) or an in vitro transcription and translation (IVTT)mixture, the IVT or IVTT mixture comprising a target molecule and adetection reagent; and (d) interrogating the IVT or IVTT mixture forreporter response, the reporter response being indicative of targetmolecule binding to the allosteric DNA-binding protein sensor and/orswitch.

In various embodiments, the allosteric DNA-binding protein sensor and/orswitch which binds to the target molecule is produced by a methodcomprising steps of (a) constructing a candidate allosteric DNA-bindingprotein sensor and/or switch, the constructing comprising (i) designinga candidate allosteric DNA-binding protein sensor and/or switch for anability to bind the target molecule, the designing optionally being insilico or (ii) undertaking directed or random mutagenesis to yield thecandidate allosteric DNA-binding protein sensor and/or switch which hasan ability to bind the target molecule; (b) providing a host cell with anucleic acid encoding the candidate allosteric DNA-binding proteinsensor and/or switch and the reporter gene system and selecting for acell comprising the candidate allosteric DNA-binding protein sensorand/or switch and the reporter gene system; (c) isolating nucleic acidsfrom the cell comprising the candidate allosteric DNA-binding proteinsensor and/or switch and the reporter gene system and contacting theisolated nucleic acids with an in vitro transcription (IVT) or an invitro transcription and translation (IVTT) mixture, the IVT or IVTTmixture comprising a target molecule and a detection reagent; and (d)interrogating the IVT or IVTT mixture by nucleic acid sequencing beforeand after selection to determine those molecules that have becomefunctionally enriched.

In various embodiments, the allosteric DNA-binding protein sensor and/orswitch which binds to a target molecule is produced by a methodcomprising steps of (a) constructing a candidate allosteric DNA-bindingprotein sensor and/or switch, the constructing comprising (i) designinga candidate allosteric DNA-binding protein sensor and/or switch for anability to bind the target molecule, the designing optionally being insilico or (ii) undertaking directed or random mutagenesis to yield thecandidate allosteric DNA-binding protein sensor and/or switch having anability to bind the target molecule; (b) contacting a solid support witha nucleic acid encoding the candidate allosteric DNA-binding proteinsensor and/or switch and selecting for a solid support comprising thecandidate allosteric DNA-binding protein sensor and/or switch; (c)isolating nucleic acids from the solid support comprising the candidateallosteric DNA-binding protein sensor and/or switch and contacting theisolated nucleic acids with an in vitro transcription (IVT) or an invitro transcription and translation (IVTT) mixture; (d) introducing areporter gene system, detection reagent, and target molecule, andinterrogating the mixture for a reporter response, the reporter responsebeing indicative of target molecule binding to the candidate allostericDNA-binding protein sensor and/or switch.

In various embodiments, the solid support is a nanoparticle and amicroparticle, a nanobead, a microbead, or an array.

In various embodiments, the allosteric DNA-binding protein sensor and/orswitch is an engineered prokaryotic transcriptional regulator familymember optionally selected from a LysR, AraC/XylS, TetR, LuxR, Lacl,ArsR, MerR, AsnC, MarR, NtrC (EBP), OmpR, DeoR, Cold shock, GntR, andCrp family member.

In various embodiments, the screening for target molecule comprises apositive or negative screen.

In various embodiments, the allosteric DNA-binding protein sensor and/orswitch is one or more of those of Table 1 and has about 1, or 2, or 3,or 4, or 5, or 10 mutations.

Any aspect or embodiment disclosed herein can be combined with any otheraspect or embodiment as disclosed herein.

DETAILED DESCRIPTION

The present invention is based, in part, on the surprising discoverythat engineered protein sensors and/or switches, such as aTFs, can bedesigned to not require cellular-based target molecule interaction andtherefore not be constrained by properties of a host cell (e.g. cellviability when contacted with a target molecule, cell transport of atarget molecule, etc.). Accordingly, the present acellular methods allowfor the development of engineered protein sensors and/or switches andthe interrogation of a wide variety of target molecules that are nototherwise available using strictly cell-based approaches.

In various embodiments, the present invention is not necessarily limitedby an inherent toxicity of the target molecules to a cell, the abilityof any target molecule to enter or remain inside screening strain cells,or the ability of any target molecule to be unaltered by cellularmachinery. Further, the present invention is not limited by the sensingof molecules either small or large, but may be extended to cellularstates such as redox potential and charge. Further, the presentinvention is not limited to the utility of allosteric transcriptionfactors that directly bind to a DNA operator, but may use effectordomains that propagate though protein cascades such as two componentsystems. Accordingly, the present methods and compositions allow formeasurable on/off response of the engineered protein that is not limitedby the ability of a cell to withstand or maintain measurableconcentrations.

In one aspect, the present invention relates to compositions and methodsfor making an engineered protein sensor and/or switch, e.g. from anallosteric protein, e.g. a transcription factor, that binds to andallows detection of a target molecule, wherein the engineered proteinsensor and/or switch is produced and screened at least in part,acellularly, and/or allows target molecules to be screened eithercellularly or acellularly.

In one embodiment, there is a provided method of making an allostericsensor and/or switch that binds controllably to a ligand different fromthat of the wild type ligand. The engineered sensor and/or switch bindsto and allows detection of the target molecule through an eliciteddetectable response wherein the engineered protein sensor and/or switchis produced and screened at least in part, acellularly, and/or allowstarget molecules to be screened acellularly not limited to methods asdescribed above.

In a further embodiment, there is a provided method of making anallosteric sensor and/or switch that binds controllably to an engineeredDNA sequence different from that of the wild type sequence in responseto the binding of a target molecule. The engineered sensor and/or switchbinds to and allows detection of a target molecule through binding to anengineered DNA sequence wherein the engineered protein sensor and/orswitch is produced and screened at least in part, acellularly, and/orallows target molecules to be screened acellularly not limited tomethods as described above.

In various embodiments, the allosteric sensor and or switch may beengineered to recognize both a new ligand as well as a new DNA bindingsite simultaneously.

In various embodiments, there is provided a method of making anallosteric DNA-binding protein sensor and/or switch which binds to atarget molecule, comprising (a) designing a candidate allostericDNA-binding protein sensor and/or switch, the DNA-binding protein sensorand/or switch being designed for an ability to bind a target moleculeand the designing optionally being in silico; (b) providing a host cellwith a nucleic acid encoding the candidate allosteric DNA-bindingprotein sensor and/or switch and a reporter gene system and selectingfor cells comprising a candidate allosteric DNA-binding protein sensorand/or switch and a reporter gene system; (c) isolating nucleic acidsfrom the cells comprising a candidate allosteric DNA-binding proteinsensor and/or switch and a reporter gene system and contacting theisolated nucleic acids with an in vitro transcription and translation(IVTT) mixture, the IVTT mixture comprising a target molecule and adetection reagent; and (d) interrogating the IVTT mixture for reporterresponse, the reporter response being indicative of target moleculebinding to the allosteric DNA-binding protein sensor and/or switch.

In some embodiments, the engineered protein sensor and/or switch, e.g.transcription factor, library members and reporter gene system reside ona single plasmid. When the plasmid is carried in a host organism, suchas E. coli or the others described herein, it is grown as singlecolonies each of which harbors a clonal library member. The reportergene and protein sensor and/or switch library members are then purifiedas plasmids and individual plasmids are introduced into an IVTT mixture(see Zubay. Ann. Rev. Genet. 1973.7:267-287, the entire contents ofwhich are hereby incorporated by reference in their entirety) to whichhas been added the target molecule and other detection reagents. After asuitable incubation period to allow expression of the reporter gene, thesolution is interrogated for reporter response.

In some embodiments, there is provided a method of making an allostericDNA-binding protein sensor and/or switch which binds to a targetmolecule, comprising (a) designing a candidate allosteric DNA-bindingprotein sensor and/or switch, the DNA-binding protein sensor and/orswitch being designed for an ability to bind a target molecule and thedesigning optionally being in silico; (b) attaching the nucleic acidencoding the candidate allosteric DNA-binding protein sensor and/orswitch to a solid support; (c) contacting the isolated nucleic acidswith an in vitro transcription and translation (IVTT) mixture, the IVTTmixture comprising a target molecule and a detection reagent; and (d)interrogating the IVTT mixture for sensor and/or switch activity in thepresence and absence of target ligand. The interrogation is notnecessarily limited to the methods described above.

In some embodiments, there is provided a method of making an allostericDNA-binding protein sensor and/or switch which binds to a targetmolecule, comprising (a) designing a candidate allosteric DNA-bindingprotein sensor and/or switch, the DNA-binding protein sensor and/orswitch being designed for an ability to bind a target molecule and thedesigning optionally being in silico; (b) generating DNA encoding theallosteric sensor and/or switch in vitro; (c) introducing the DNA into adisplay system—for example but not limited to ribosome display, mRNAdisplay, phage display, cell display—(d) interrogating the displayedsensors for DNA binding in the presence and absence of ligand. Theinterrogation being indicative of activity. This reporter-free strategyto evaluate sensors is facilitated by coupling the sensor protein to themRNA transcript encoding its translation, either by association withstalled ribosomes as in ribosome display (see Hanes, et al. PNAS. 1997;94(10):4937-4942.) or through covalent linkage as in mRNA display (seeWilson et al. PNAS. 2001; 98(7):3750-5). Coupling of genetic sequence tothe functional protein allows for faster identification of functionalsensor sequences, and rapid cycles of in vitro selection, mutation andevolution of the sensor proteins. Because many aTF sensors operate asobligate homodimers, this strategy is further facilitated by creating a‘dimeric’ single chain sensor with a linker sequence that allows properfolding (see Krueger et al. Nucleic Acids Research. 200331(12):3050-3056) from an initial mutant monomer sensor gene throughPCR, ligation, transposon/transposase system, recombinase, CRISPR/Cas9,or combination of these methods. In this way, both dimers encode thesame monomer sequence, as a single chain that would greatly favorhomodimerization rather than heterodimerization of different mutantswithin a large mutant pool. In some embodiments, the engineered monomeris coupled to a wild type monomer to create a heterodimeric singlechain.

In other embodiments, sensors are assayed by their affinity for anoperator DNA sequence, without a separate reporter being expressed,and/or by a change in this operator DNA affinity in the presence of atarget chemical.

For example, this allows a pool of sensors to be evaluated initially forDNA binding capability in the absence of a target chemical by capture onimmobilized DNA operator sequences (e.g. on beads, microarray chips,microfluidic device, flow cell, chromatography column), and thensecondly evaluated for response to a target chemical by release from theimmobilized DNA operator sequences. This reporter-free strategy toevaluate sensors is facilitated by coupling the sensor protein to themRNA transcript encoding its translation, either by association withstalled ribosomes as in ribosome display (see Hanes, et al. PNAS. 1997;94(10):4937-4942.) or through covalent linkage as in mRNA display (seeWilson et al. PNAS. 2001; 98(7):3750-5). Coupling of genetic sequence tothe functional protein allows for faster identification of functionalsensor sequences, and rapid cycles of in vitro selection, mutation andevolution of the sensor proteins. Because many aTF sensors operate asobligate homodimers, this strategy is further facilitated by creating a‘dimeric’ single chain sensor with a linker sequence that allows properfolding (see Krueger et al. Nucleic Acids Research. 200331(12):3050-3056) from an initial mutant monomer sensor gene throughPCR, ligation, transposon/transposase system, recombinase, CRISPR/Cas9,or combination of these methods. In this way, both dimers encode thesame monomer sequence, as a single chain that would greatly favorhomodimerization rather than heterodimerization of different mutantswithin a large mutant pool. In some embodiments, the engineered monomeris coupled to a wild type monomer to create a heterodimeric singlechain. In some embodiments, the engineered protein sensor and/or switch,such as an aTF, and nucleic acids comprising the aTF in addition to acandidate reporter gene system contacting an in vitro transcription andtranslation (IVTT) mixture and detection reagent results in thegeneration of a reporter protein upon ligand binding. For example, insome embodiments, the engineered protein sensor and/or switch, such asan aTF, is contacted with a target molecule and a reporter is generatedusing an acellular method, e.g. IVTT. The reaction mixture can then beinterrogated by the reporter response where the reporter response isindicative of target molecule binding to the allosteric DNA-bindingprotein sensor and/or switch.

In another aspect, the present invention relates to compositions andmethods for detecting a target molecule, optionally cellularly oracellularly, using an engineered protein sensor and/or switch, such asan aTF, which is produced with cellular or acellular methods, such as invitro transcription and translation (IVTT) as described herein. Invarious embodiments, the detection is acellularly, e.g. by employingmethods such as in vitro transcription and translation (IVTT) to detecta reporter that is allosterically linked to the engineered proteinsensor and/or switch, such as an aTF. In some embodiments, theengineered protein sensor and/or switch, such as an aTF, can optionallybe produced acellularly or within the cell. In some embodiments, theengineered protein sensor and/or switch, such as an aTF, detects targetmolecule binding via acellular methods, for instance the production of adetectable reporter via an acellular method, e.g. IVTT.

Useful reporters include proteins with unique spectral signatures, suchas, without limitation, green fluorescent protein whose expression maybe determined using a microtiter plate fluorimeter, visual inspection,or a fluorescence activated cell sorter (FACS). Reporters also include,without limitation, spectral signatures based on absorbance, physicalproperties such as magnetism and impedance, changes in redox state,assayable enzymatic activities, such as a phosphatase,beta-galactosidase, peroxidase, luciferase, or gas generating enzymes.Alternatively, a linear single or double stranded DNA that encodes thereporter and transcription factor library member may be used as areporter in cases not limited to amplification by polymerases.

The present invention includes a reporter gene system, which comprises aprotein having a unique spectral signature and/or assayable enzymaticactivity. Illustrative reporter systems detection methods include, butare not limited to, those using chemiluminescent or fluorescentproteins, such as, for example, green fluorescent protein (GFP),enhanced green fluorescent protein (EGFP), Renilla Reniformis greenfluorescent protein, GFPmut2, GFPuv4, yellow fluorescent protein (YFP),enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein(CFP), enhanced cyan fluorescent protein (ECFP), enhanced bluefluorescent protein (EBFP), chromoproteins, citrine and red fluorescentprotein from discosoma (dsRED), infrared fluorescent proteins,luciferase, umbelliferone, rhodamine, fluorescein,dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, andthe like. Examples of detectable bioluminescent proteins include, butare not limited to, luciferase (e.g., bacterial, firefly, click beetleand the like), luciferin, aequorin and the like. Examples of detectableenzyme systems include, but are not limited to, galactosidases,glucorinidases, phosphatases, peroxidases, cholinesterases, proteases,and the like. In certain other embodiments, the reporter systemsdetection methods include an enzyme. In certain other embodiments, thedetectable marker is a non-essential gene that can be assayed rapidlyfor genetic variation by qPCR. In certain other embodiments, thedetectable marker is a drug resistance marker that can be readilyassessed for functionality by reverse selection. In some embodiments,the detectable marker is a nutritional marker, e.g. production of arequired metabolite in an auxotrophic strain, ability to grow on a solecarbon source, or any other growth selection strategy known in the art.

In certain embodiments, the reporter is composed of two or morecomponents which when present together produce the functional reporter.Examples include split GFPs, and enzymes such as luciferase, betagalactosidase, beta lactamase, and dihydrofolate reductase. One or morecomponents of a split reporter may be introduced exogenously allowingdetection of cellular production of fewer components. The split reportermay be can be used to detect split reporter-fused to another proteinallowing detection either inside the cell, outside the cell, or both.For instance, a split GFP fusion protein may be excreted by a cellencapsulated with the complementing reporter component such that theproducing cell does not have the capacity to produce a functionalreporter until encapsulated with its complement. One or more componentsof such a split systems may be produced independently and added as adetection reagent to the cells being assayed.

For example, beta-glucosidase and Antarctic phosphatase may be used asreporter systems with their corresponding fluorogenic substratesfluorescein di-(p-D-glucopyranoside) and fluorescein diphosphate (FIG.7, FIG. 8).

In some embodiments, the binding event of the aTF itself is utilized topresent a physical readout of aTF state through either optical ornonoptical methods in an acellular environment. For example in anon-limiting manner, the aTF is linked to a fluorescent protein and theDNA binding site is linked to a quencher molecule. Fluorescent readoutis possible only when the aTF is released from the DNA binding siteitself. This method allows for a direct readout of aTF binding events.This strategy is not limited to fluorophore quencher pairs, but may alsoemploy other read outs such as split proteins. Additionally, the bindingevent may be used to physically separate functional proteins fromnon-functional proteins in the case of protein display methods.

In some embodiments, the engineered protein sensor and/or switch, suchas an aTF, detects target molecule binding via acellular methods, forinstance by controlling the activity of a polymerase that directlyamplifies the genotype of the functional sensor and/or switch. Thepolymerase may either be a DNA or RNA polymerase that either amplifiesthe RNA and/or DNA versions of the genotype (FIG. 2, FIG. 3, FIG. 4).

In various embodiments, the present methods include various detectiontechniques, e.g. for reporter signal. Such detection techniques mayinvolve a microscope, a spectrophotometer, a fluorometer, a tubeluminometer or plate luminometer, x-ray film, magnetic fields, ascintillator, a fluorescence activated cell sorting (FACS) apparatus, amicrobial colony picker (e.g., QPix), a microfluidics apparatus, abead-based apparatus or the like.

In some embodiments, strains engineered for protein secretion may beassayed for secretion by fusing a split reporter, such as GFP, to thesecreted protein and assaying in cell-free compartments.

Useful cell-free compartments include without limitation standard growthfermenters, flasks, culture tubes, and plastic ware, microliter plates,patterned microwells, microdroplets generated either in bulk (FIG. 2) ormicrofluidically (FIG. 3 and FIG. 4).

Bulk emulsions may be formed without limitation using the BioRad dropletoil for supermixes or a suitable such as mineral oil and span 80 orfluorinated oils such as HFE7500 and a fluorinated surfactant (FIG. 2,panel a). Droplet diameters may range from 1 um to 500 um withoutlimitation. Microfluidic emulsions may be formed without limitationusing commercial fluorophilic chips or house-made PDMS chips with ahydrophobic surface with the HFE7500 fluorinated oil and Dolomite'sproprietary PicoSurf surfactant (FIG. 3). Other commercially availablechips, oils, and surfactants may be used as well as noncommercial chipsand oil-surfactant mixes. A single commercial Dolomite chip can producedroplets at a rate of <20 kHz allowing the production of up to 576, 000,000 droplets from a single chip in a single workday. This assay isamenable to parallel droplet formation for improved throughput.Microfluidically generated water-in-oil emulsions are monodisperse andstable for >7 days at 37° C. Droplet diameters may range from 1 um to500 um without limitation. Droplets may also be generated inside oftubes using an air interface to separate droplets. In this strategy,both pressures and atmospheric compositions may be controlled inside ofthe droplets.

In some embodiments, water-in-oil droplets may be utilized tocompartmentalize a single E. coli cell overexpressing a unique aTF (FIG.2, panel a, FIG. 3, panel a, and FIG. 5, panel a). The librarydiversity—in other words the number of unique E. coli capable of beingscreened—is limited to the number of droplets produced. Each compartmentalso contains the effector (ligand) of interest regulating aTF activity,a polymerase reporter gene or promoter upstream of the aTF gene underthe control of the aTF operator site, IVT or IVTT reagents as needed, aswell as a chemical or enzymatic lytic agent.

In one embodiment, a pool of E. coli containing a library of engineeredaTFs are encapsulated and the E. coli cells are lysed releasing theaTFs. aTFs bound to the operator site directly upstream a reporter DNApolymerase gene that prevents IVTT of the reporter polymerase. Any DNApolymerase may be used. aTFs responsive to the effector of interestrelease the DNA allowing IVTT of the polymerase. aTFs unresponsive tothe effector of interest repress IVTT and therefore production of thepolymerase (FIG. 4). Afterwards, droplets are immediately amplified byPCR using aTF specific primers. Functional aTFs that produce morepolymerase are enriched over their non-functional counterparts (FIG. 2,panel b). Amplicons are cloned into the correponding expression vector,transformed back into the E. coli strain, and plated on solid support.Colonies may be scraped from the solid support and grown in liquid forsubsequent rounds of enrichment or colonies or picked as isolates forscreening. Unique colonies containing functional aTF genes picked fromplates may be tested for activity. Activity is tested with lysate in 96or 384 well blocks using fluorescent assays or using microfluidicdroplet-based assays.

In a second embodiment, a pool of E. coli containing a library ofengineered aTFs are encapsulated and the E. coli cells are lysedreleasing the aTFs. aTFs bound to the operator site directly upstream ofthe aTF gene prevent transcription by an RNA polymerase. Any RNApolymerase may be used. aTFs responsive to the effector of interestrelease the DNA allowing RNA transcription of the aTF gene. aTFsunresponsive to the effector of interest repress transcription andtherefore amplification of the aTF genotype (FIG. 4). Subsequentbreaking of the droplets and recovery of the RNA followed by RT-PCR withaTF specific primers rapidly amplifies the genotype of functional aTFs.Amplicons are cloned into the correponding expression vector,transformed back into the E. coli strain, and plated on solid support.Colonies may be scraped from the solid support and grown in liquid forsubsequent rounds of enrichment or colonies or picked as isolates forscreening. Unique colonies containing functional aTF genes picked fromplates may be tested for activity. Activity is tested with lysate in 96or 384 well blocks using fluorescent assays or using microfluidicdroplet-based assays.

In some embodiments, a method of the invention comprisesmicroencapsulating an individual cell, e.g. a bacterium, hosting thelibrary plasmid with lysis reagent, IVTT mixture, the target molecule,and, if using a reporter enzyme system, substrate using one or moremicrofluidic devices (see Zinchenko, et al. Analytical Chem.2014.86:2526-2533 and A. Fallah-Araghi, et al. Lab Chip. 2012. 12:882-891, the contents of which are hereby incorporated by reference intheir entireties). Following conditions suitable for cell lysis themicrodroplets are incubated at conditions suitable for IVTT. They arethen incubated for the appropriate time to develop the reporter protein.Finally, library members which produce a desired response are isolatedfrom those that do not using a microfluidic device or FACS (FIG. 5,panels a and b). The plasmids of the positive responders are purifiedfrom the microdroplets, amplified and re-transformed into host bacteriumfor sequencing and clonal functional testing as described herein.

In various embodiments sensors and/or switches may be screened for adesired activity inside of water-in-oil-in-water emulsions. Thewater-in-oil emulsions are formed microfluidically.

Microfluidic double emulsions may be formed using Dolomite commercialchips or house-made PDMS chips with the HFE7500 fluorinated oil andDolomite's proprietary PicoSurf surfactant (FIG. 5, panels c-e). Othercommercially available chips, oils, and surfactants may be used as wellas noncommercial chips and oil-surfactant mixes.

Double emulsions may be formed in two steps (FIG. 5, panels c-e). Thefirst emulsion is made using a Domolomite commercial fluorophilic chipand the second is with a hydrophilic chip. Alternatively PDMS chipssufficiently oxidized to have a hydrophobic surface may supplement thefirst Dolomite chip while a newly plasma treated chip with a hydrophiliccoating may replace the second chip. Alternatively, the PDMS chip may betreated with PVA or an alternative reagent to bear a semi-permanenthydrophilic surface. A single commercial Dolomite chip can producedroplets at a rate of <20 kHz allowing the production of 576, 000, 000droplets from a single chip in a single workday. This assay is amenableto parallel droplet formation. Microfluidically generated water-in-oilemulsions are monodisperse and stable for >7 days at 37° C. The secondemulsification takes place at half the rate to prevent droplet shearing.Double emulsions are stable for >7 days at 37° C.

Double emulsions may also be produced in a single step using custom PDMSchips (FIG. 6). In this aspect, the first emulsion directly precedes theformation of the second emulsions on the same chip. This processcircumvents the need to produce two independent emulsions but proceedsat a rate 25% that of the single emulsion step.

In both of these cases, water-in-oil-in-water droplets are utilized tocompartmentalize a single E. coli cell overexpressing a unique aTF (FIG.5, panel a). The number of unique E. coli capable of being screened islimited to the amount of unique droplets being made. Inside of eachcompartment is also the effector (ligand) of interest regulating aTFactivity, a reporter enzyme gene under the control of the aTF operatorsite, a fluorogenic substrate as described above, additional lysate asneeded, as well as a chemical or enzymatic lytic agent.

Once encapsulated, the E. coli cells are lysed releasing the aTFs. aTFsbind to the operator site preventing the expression of the reporterenzyme. aTFs responsive to the effector of interest release the DNAallowing expression of the reporter enzyme. aTFs unresponsive to theeffector of interest repress reporter enzyme expression. As the reporterenzyme is expressed, the enzyme converts the substrate from anon-detectable to a detectable state (FIG. 5, panel b).

Droplets containing functional aTFs will allow for the production ofsufficient signal to enable separation using a suitable method. Oncesorted, plasmids encoding the functional aTFs are transformed into thedesired E. coli strain and plated. Colonies are scraped from the plateand grown in liquid for subsequent rounds of screening.

Unique colonies containing functional aTF genes may be picked fromplates and tested for activity. Activity is tested with lysate in 96 or384 well blocks using fluorescent assays or using microfluidically-basedassays.

As an example, in some embodiments, a lytic plasmid contains areplication origin (e.g. ColE1), selectable marker (e.g. AMP), an IVTTtranscribable (e.g. T7) reporter gene (e.g. alkaline phosphatase, AP)under control of the design aTF operator (e.g. TetO), Lacl, lacOcontrolled lytic system (e.g. T4 holin and T4 lysozyme), and the designaTF (e.g. TetR). A library of aTF designs is created and transformedinto an E. coli strain which has no IVTT (e.g. T7) polymerase and inwhich alkaline phosphatase is deleted from the genome. A growing cultureof these bacteria is washed in a buffer and passed through amicrofluidic device at conditions which encapsulated a reagent streamand, on average, about one bacterium per microdroplet. The reagentstream includes IPTG (without wishing to be bound by theory, to induceself lysis by the lytic system), IVTT reagents, the target moleculeagainst which the aTF is designed, and the AP substrate fluoresceindiphosphate (FDP). After microencapsulation, the microdroplets areencapsulated in a second microfluidic device to produce a population ofwater stable double emulsion microdroplets. The microdroplets are thenincubated to allow bacterial lysis, transcription and translation of APif the TetR library member recognizes target molecule, and developmentof fluorescein from the FDP. Bright (positive) microdroplets are sortedfrom dim microdroplets using a FACS machine. The pool of positivedroplets is dissolved with an organic solvent to release the contents ofthe positive droplets and the mixture of positive TetR genes areamplified using PCR. The positive TetR mixture is then cloned back intothe plasmid backbone and retransformed into the host E. coli strain, andgrown under selection. This positive library is then put through theprocess again to confirm the results, with the possibility to alter theconcentration of the target molecule to identify more or less sensitivelibrary members (FIG. 5). Recovered confirmed positive plasmids are thenagain amplified, cloned, and transformed into the host strain and grownclonally. Clonal sensor plasmids are then characterized once again, forinstance by repetition of the present microencapsulation system or othertechniques (e.g. to measure in bulk), by looking at their response to arange of concentrations of the target molecule. The sequence of TetRclones with the desired properties are then determined. The targetsensor can then be cloned into its working context for strainoptimization or genome engineering or other downstream use.

In various embodiments, lysis may also be effected using an induciblecell lytic system encoded on the host cell genome, a separate plasmid,or encoded on the library plasmid itself (Morita, et al. Biotechnol.Prog. 2001. 17(3):573-6, the entire contents of which are herebyincorporated by reference). In such a system, the lysis inducer isincluded with the IVTT mixture, target molecule, and other requiredsubstrates depending on the reporter system being used. Inducible lyticsystems often include one or more phage proteins such as, for example,psi X174 E protein (Henrich Mol. Gen. Genet. 1982. 185(3)493-7, theentire contents of which are hereby incorporated by reference or T4holin and lysozyme.

Microfluidic chip designs are not limited to those presented above. Insome embodiments, double emulsions are generated in one step (FIG. 6).For example, chips made with PDMS and external aqueous phase channelstreated with 1% PVA, may be used to form double emulsion droplets in asingle step with the HFE7500 and fluorinated surfactant. Other oil andsurfactant combinations may be utilized. In this previously publishedchip design [Nie Joumal of the American Chemical Society 2005. 127.8058-63], the internal aqueous phase is coflowed with 2 streams ofoil-surfactant in which they are encapsulated by the external aqueousphase. This chip design results in both droplets being formed in asingle pinch-flow interface. In some embodiments, individual sensorsmight be assayed by enclosing each one in emulsion-type droplets. Forexample, this may be facilitated by merging two or more droplets ortypes of droplets (e.g. containing different DNA sequences or enzymes orchemicals) in a microfluidic device. Droplets may also be assessed andsorted on-chip using techniques like but not limited fluorescenceactivated droplet sorting (FADS) or absorbance activated droplet sorting(AADS).

In some embodiments, a cell or cells hosting the cellularly oracellularly derived sensor system is coencaspulated with a metabolicallyengineered cell or cells, or “producing strain, ” having been engineeredby one or more of the methods described herein, designed to produce thetarget molecule capable of being detected by the sensor system. This isuseful, inter a/ia, if the producing strain constitutively exports thesensed molecule into its growth medium creating the case where a highproducing and low producing strain both have the same intracellularconcentration of the molecule of interest but the medium of the highproducing strain has a greater concentration. In such cases, thedetector strain may be used to discern high from low producers. In otherembodiments, the present invention includes the use of multiple dropletscontaining whole or lysed cells from different hosts. For instance, insome embodiments, a first droplet comprises whole or lysed cells with anengineered sensor while a second droplet comprises whole or lysed cells,“producer strains”, with the target molecule (e.g. host cells that areengineered to produce a target molecule as described elsewhere herein).For example, in some embodiments, the first droplet comprising whole orlysed cells with an engineered sensor is used to detect production of atarget molecule in a different host (in the form of whole or lysed cellsin a droplet). As such, inter a/ia, this permits detection of the targetmolecule at levels that are beyond what could be undertaken if theengineered sensor were present solely in the host cells that areengineered to produce a target molecule. In some embodimentstranscription/translation of the sensor and/or the reporter it controlsare driven by in vitro transcription and translation (IVTT), asdescribed in Zubay. Ann. Rev. Genet. 1973.7:267-287, the entire contentsof which are hereby incorporated by reference in their entirety or TX-TLas described in Shin and Noireaux, J Biol. Eng. 4, 8 (2010) and USPatent Publication No. 2016/0002611, the entire contents of which arehereby incorporated by reference in their entireties. Microencapsulationof single producers, either harboring the sensor machinery orcoencapsulated with sensor cells, is also a useful technique in caseswhere the molecule is highly diffusible across the cell membrane, makingscreening in batch liquid culture impossible.

In other embodiments, cells are lysed in one microdroplet which is thenmerged with a second microdroplet containing the reagents required forIVT or IVTT.

In another embodiment, DNA encoding a single sensor library member iscaptured on a bead and encapsulated in a microdroplet (see Dressman, etal. PNAS 2003 100(15):8817-8822), such that it may be amplified and/orexpressed through IVTT. The droplet is then merged with reporterreagents for response interrogation. This may be beneficial when the aTFis not expressible and/or expressed in a functional state in suitablescreening systems.

In another embodiment, DNA encoding a reporter gene is captured on abead and encapsulated in a microdroplet, such that it may be amplifiedand/or expressed through IVTT. The droplet is then merged with reporterreagents for response interrogation.

In another embodiment, the transcription factor library resides on oneplasmid while the reporter gene system resides on a second plasmid. Byhaving two separate plasmids, the effective concentration of reportergene to sensor library members may be adjusted to facilitateidentification of active library members. This is useful where simplyusing higher versus lower promoter strength is not enough control, forinstance.

In another embodiment, the reporter system is encoded in the hostgenome.

In another embodiment, the DNA encoding the reporter is present only inthe droplet containing the reagents required for IVT or IVTT, and theDNA encoding the sensor is present in the other droplet.

In another embodiment, the DNA encoding the sensor is present only inthe droplet containing the reagents required for IVT or IVTT, and theDNA encoding the reporter is present in the other droplet.

In other embodiments, the present invention includes the use of multipledroplets containing whole or lysed cells from different hosts. Forinstance, in some embodiments, a first droplet comprises whole or lysedcells with an engineered sensor while a second droplet comprises wholeor lysed cells with the target molecule (e.g. host cells that areengineered to produce a target molecule as described elsewhere herein).For example, in some embodiments, the first droplet comprising whole orlysed cells with an engineered sensor is used to detect production of atarget molecule in a different host (in the form of whole or lysed cellsin a droplet). As such, inter alia, this permits detection of the targetmolecule at levels that are beyond what could be undertaken if theengineered sensor were present in the host cells that are engineered toproduce a target molecule.

In some embodiments, the present methods are designed to delay thecreation of the reporter message relative to the designed aTF; anapproach which enables concentrations of the designed aTF protein toreach the level required to repress transcription of the reporter. Forexample, the reporter transcription is controlled by two repressorswhich recognize separate operator sites on the reporter gene's promoterregion. The reporter transcription is thus suppressed in the IVTT systemuntil both transcription factors bind an inducing molecule. Thispermits, inter a/ia, delaying transcription of the reporter messageuntil a sufficient concentration of the engineered aTF is built up inthe IVTT mix to allow detection of its response to its non-cognatetarget ligand. For instance, in some embodiments, a library of TetRdesigns is produced with candidate designs to alter ligand specificityfrom, e.g., tetracycline to a target molecule, such as curcumin. TheTetR gene is driven by a promoter recognized by the IVTT but not thehost cell, such as T7. The reporter gene is similarly driven by apromoter recognized by the IVTT and modulated by both Tetracyline andLacl operators (TetO and LacO in FIG. 1). When bacteria harboring such asystem are lysed and mixed with the IVTT system, reporter transcriptionis halted, initially, by the wild type constitutively expressed Laclonly (“Initial State” in FIG. 1). As IVTT proceeds, concentrations ofthe engineered TetR increase to where it may also repress the reporter(“Intermediate State” in FIG. 1). At this time, an inducer molecule ofLacl may be added to interrogate if the curcumin has preventedengineered TetR binding its operator. This description is made by way ofexample and is equally applicable to other aTFs and target molecules.

In various embodiments, the present methods are extended to include anysubstrate that is changed into an inducing molecule, for example IPTG inthe case of Lacl, whose concentration is gradually increased through anenzymatic activity. One advantage of this approach is that a singlemixture containing the non-cognate target ligand, substrate, IVTT, andlysis system reduces the number of components that need to be combined.In its final state, the multiple repressor system only allows thecreation of reporter message and thus reporter protein when theengineered protein candidate is modulated by the non-cognate targetligand.

Similarly, the effective concentration of the non-engineered repressormay be lowered by targeted degradation, by, for example, proteases.Additionally or alternatively, in various embodiments, thenon-engineered repressor may be sensitive to additional treatments. Forexample, it may denature or become inactive when, for example, one ormore of temperature, pH, ionic strength, and charge, is altered (e.g.raised or lowered). Additionally or alternatively, in variousembodiments, the non-engineered repressor may be sensitive to additionaltreatments, such that it denatures or becomes inactive when in thepresence of light.

In other embodiments, the reporter message may be made to be unstable inthe absence of a stabilizing agent, whereupon the stabilizing reagent isadded either together with or subsequent to the addition of the IVTTand/or lysis reagents.

In other embodiments, a rapidly degrading reporter can be utilized toenhance the distinction between the response range of sensors that areresponding to the target molecule.

In another aspect, the present invention relates to compositions andmethods for detecting, optionally acellularly, a target molecule usingan engineered protein sensor and/or switch, such as an aTF, which isoptionally detected for the desired functionality with acellularmethods, such as in vitro transcription (IVT) or in vitro transcriptionand translation (IVTT) as described herein. For instance, in someembodiments, the detection of a target molecule is in a cell, such asany of those described herein, which has been manipulated to produce thetarget molecule.

In some aspects, the present invention allows for engineering or use ofa protein sensor and/or switch for which the protein sensor and/orswitch's natural promoter and/or operator does not function suitably ina host cell. In some embodiments, the invention provides transfer of afunctional operator site from one organism to another. For instance,such transfer is applicable to the present cell-free senor engineeringas described herein and the use of an engineered sensor in a host cell(e.g. to detect production of a target molecule). In some embodiments,e.g. when deploying the present sensors (e.g. to detect production of atarget molecule in a host cell), the present invention allows for theintroduction of protein sensors and/or switches, e.g. aTFs, from avariety of organisms and the operation of the present sensing in avariety of host organisms, including those particularly desired formetabolic engineering, such as any of the host cells described herein.

An illustrative method to transfer a functional operator site from oneorganism to another, such organisms may be selected from the cellsdescribed herein, is to clone the intergenic region immediately upstreamof a gene regulated by the protein sensor and/or switch, e.g. aTF, ofinterest immediately upstream of reporter gene that is carried in thedesired host organism. This naïve approach assumes that thetranscriptional promoter will also function in the host organism.Assuming no host repressors recognize the exogenous operator site oncecloned, the reporter will be constitutively on until expression of theregulator protein in a mode to bind its operator and repress thereporter signal. The basic approach has the advantage of, among others,not needing any information about the actual DNA sequence of theoperator site but may suffer from the fact that the intergenic regioncloned may have a promoter region incompatible with the new hostorganism.

To circumvent the problem of the host cell not being able to utilize theforeign promoter, an operator sequence may be cloned into a promoterregion known to function in the host organism between thetranscriptional promoter and ribosome binding site. Sometimes operatorsequences are longer than the allowable sequence space between thepromoter and RBS sites. In such cases the operator may be placed 5′ or3′ to the promoter site. In some cases, the operator consists of tworegions of DNA separated by some number of bases. In such cases, it maybe advantageous to flank either or both the promoter and/or RBS sitewith the operator binding sequence. In some cases, multiple sets ofoperator sites may by introduced in the promoter RBS region to increasethe number of binding aTFs to more than 1.

Construction of synthetic promoter/operators allow the aTF to functionin any organism for which the promoter/RBS paradigm is maintained,including eukaryotes such as yeast. Optionally, in eukaryotes, the aTFmay be expressed as a fusion with a nuclear localization signal. Thesynthetic promoter/operators also function in the context of IVTT solong as the promoter and RBS are recognized by the IVTT system. RBS maybe replaced by internal ribosome entry sites for translation initiation.

In various embodiments, the present invention allows for engineering ahost cell to produce a target molecule and the target molecule isdetected or detectable using one or more of the engineered proteinsensor and/or switch. In various embodiments, cells are engineered witha multiplex genome engineering technique (e.g.

Multiplexed Automated Genome Engineering (MAGE, see, e.g., Wang et al.,Nature, 460:894-898 (2009); Church et al., U.S. Patent No. 8,153,432,the contents of which are hereby incorporated by reference in theirentireties), conjugative assembly genome engineering (CAGE, see, e.g.,Isaacs, F. J. et al. Science 333, 348-353, the contents of which arehereby incorporated by reference in their entireties), a methodinvolving a double-strand break (DSB) or single-strand break or nickwhich can be created by a site-specific nuclease such as a zinc-fingernuclease (ZFN) or TAL effector domain nuclease (TALEN) or BurrH bindingdomain (BuD)-derived nucleases, or CRISPR/Cas9 system with an engineeredcrRNA/tracrRNA (or synthetic guide RNA) to guide specific cleavage (see,e.g., U.S. Patent Publications 2003/0232410; 2005/0208489; 2005/0026157;2005/0064474; 2006/0188987; 2009/0263900; 2009/0117617; 2010/0047805;2011/0207221; 2011/0301073 and International Patent Publication WO2007/014275, and Gaj, et al. Trends in Biotechnology, 31(7), 397-405(2013), the contents of which are hereby incorporated by reference intheir entireties, or utilizes the organism's native CRISPR systemtogether with a recombinase (e.g. ssDNA recombinase system, which mayinclude a single-stranded annealing protein (SSAP), such as the A Redrecombineering system (e.g., Beta protein) or RecET system (e.g., recT),or homologous system, including Rad52-like (of which A Red Beta, Sak,and Erf are members), Rad51-like (e.g., Sak4), and Gp2.5-like, each withdistinct sequence profiles and folds. Datta et al., PNAS USA,105:1626-31 (2008); Lopes, A., Nucleic Acids Research, 38(12),3952-3962, which are hereby incorporated by reference in theirentireties, see also International Patent Publication WO/2015/017866,the contents of which are hereby incorporated by reference in itsentirety), the disclosures of which are incorporated by reference intheir entireties for all purposes)).

In various embodiments, the engineered protein sensor and/or switch isan aTF, for instance a eukaryotic aTF. In various embodiments,engineered protein sensor and/or switch is an engineered version of aprokaryotic transcriptional regulator family such as a member of theLysR, AraC/XylS, TetR, LuxR, Lacl, ArsR, MerR, AsnC, MarR, NtrC (EBP),OmpR, DeoR, Cold shock, GntR, and Crp families.

In various embodiments, engineered protein sensor and/or switch is anengineered version of a prokaryotic transcriptional regulator familysuch as a member of the AbrB, AlpA, AraC, ArgR, ArsR, AsnC, BetR, Bhl,CitT, CodY, ComK, Crl, Crp, CsoR, CtsR, DeoR, DnaA, DtxR, Ecf, FaeA,Fe_dep_repress, FeoC, Fis, FlhC, FlhD, Fur, GntR, GutM, Hns, HrcA, HxIR,IcIR, KorB, Lacl, LexA, Lsr2, LuxR, LysR, LytTR, MarR, MerR, MetJ, Mga,Mor, MtIR, NarL, NtrC, OmpR, PadR, Prd, PrrA, PucR, PuR, Rok, Ros_MucR,RpiR, RpoD, RpoN, Rrf2, RtcR, Sarp, SfsA, SinR, SorC, Spo0A, TetR, TrmB,TrpR, WhiB, Xre, YcbB, and YesN families.

In various embodiments, engineered protein sensor and/or switch is anengineered version of a member of the TetR family of receptors, such asAcrR, ActIl, AmeR AmrR, ArpR, BpeR, EnvR E, EthR, HydR, IfeR, LanK,LfrR, LmrA, MtrR, Pip, PqrA, QacR, RifQ, RmrR, SimReg, SmeT, SrpR, TcmR,TetR, TtgR, TtgW, UrdK, VarR, YdeS, ArpA, Aur1B, BarA, CalR1, CprB,FarA, JadR, JadR2, MphB, NonG, PhIF, TyIQ, VanT, TarA, TyIP, BM1P1,Bm1P1, Bm3R1, ButR, CampR, CamR, CymR, DhaR, KstR, LexA-like, AcnR,PaaR, Psbl, ThIR, UidR, YDH1, Betl, McbR, MphR, PhaD, Q9ZF45, TtK, Yhgdor YixD, CasR, IcaR, LitR, LuxR, LuxT, OpaR, Orf2, SmcR, HapR, Ef0113,HlylIR, BarB, ScbR, MmfR, AmtR, PsrA, and YjdC.

The engineered protein sensor and/or switch may be an engineered versionof a two-component or hybrid two-component system that directly bindboth a ligand and DNA or work through a protein cascade.

In various embodiments, the engineered protein sensor and/or switch isan aTF, for instance a eukaryotic aTF. In various embodiments,engineered protein sensor and/or switch is an engineered version of RovM(Yersinia pseudotuberculosis), HcaR (Acinetobacter), BIcR (Agrobacteriumtumefaciens), HetR (Anabaena spp.), HetR (Anabaena spp.), DesR (B.subtilis), HyllIR (Bacillus cereus), PlcR (Bacillus cereus), CcpA(Bacillus megaterium), YvoA (Bacillus subtilis), AhrR (Bacillussubtilis), MntR (Bacillus subtilis), GabR (Bacillus subtilis), SinR(Bacillus subtilis), CggR (Bacillus subtilis), FapR (Bacillus subtilis),OhrR (Bacillus subtilis), PurR (Bacillus subtilis), Rrf2 (Bacillussubtilis), BmrR (Bacillus subtilis), CcpN repressor (Bacillus subtilis),TreR (Bacillus subtilis), CodY (Bacillus subtilis), yfiR (Bacillussubtilis), OhrR (Bacillus subtilis), Rex (Bacillus subtilis, Thermusthermophilus, Thermus aquaticus), NprR (Bacillus thuringiensis), BtAraR(Bacteriodes thetaiotaomicron), AraR (Bacteroides thetaiotaomicron VPI),DntR (Burkholderia cepacia), CmeR (Camplylobacter jejuni), CviR(Chromobacterium violaceum), TsaR (Comamonas testosteroni), CGL2612(Corynebacterium glatamicum), CIgR (Corynebacterium glutamicum), LIdR(CGL2915) (Corynebacterium glutamicum), NtcA (Cyanobacterium Anabaena),HucR (Deinococcus radiodurans), Lacl (E. coli), PrgX (Enterococcusfaecalis), NikR (Helobacter pylori), LmrR (Lactococcus lactis), CcpA(Lactococcus lactis), MtbCRP (Mycobacterium tuberculosis), EthR(Mycobacterium tuberculosis), MosR (Mycobacterium tuberculosis), PhoP(Mycobacterium tuberculosis), Rv1846c (Mycobacterium tuberculosis), EthR(Mycobacterium tuberculosis), LysR (Neisseria meningitdis), NMB0573/AsnC(Neisseria meningitidis), TetR-class H (Pasteurella multocida), MexR(Pseudomonas aeruginosa), DNR (Pseudomonas aeruginosa), PA01(Pseudomonas aeruginosa), PA2196 (Pseudomonas aeruginosa), ttgR(Pseudomonas putida), Cra (Pseudomonas putida), QscR (Psudemonasaeruginosa), ActR (S. coelicolor), SCO0520 (S. coelicolor), CprB (S.coelicolor), SlyA (Salmonella enterica SlyA), FapR (Staphylococcusaureus), QacR (Staphylococcus aureus), SarZ (Staphylococcus aureus),IcaR (Staphylococcus aureus), LcaR (Staphylococcus epidermidis), SMET(Stenotrophomonas maltophilia), PcaV (SCO6704) (Streptomycescoelicolor), SCO4008 (Streptomyces coelicolor), NdgR (Streptomycescoelicolor), CprB (Streptomyces coelicolor), SCO0253 (Streptomycescoelicolor), TetR family (Streptomyces coelicolor), SCO0520(Streptomyces coelicolor), SCO4942 (Streptomyces coelicolor), SCO4313(Streptomyces coelicolor), TetR family (Streptomyces coelicolor),SCO7222 (Streptomyces coelicolor), SCO3205 (Streptomyces coelicolor),SCO3201 (Streptomyces coelicolor), ST1710 (Sulfolobus tokodaii ST1710),HrcA (Thermotoga maritima), TM1030 (Thermotoga maritime), tm1171(thermotoga maritime), IcIR (thermotoga maritime), CarH (Thermusthermophilus), FadR (Vibrio cholerae), SmcR (Vibrio vulnificus), andRovA (Yersinia pestis).

In various embodiments, engineered protein sensor and/or switch is anengineered version of MphR, AlkS, AlkR, CdaR, BenM, RUNX1, MarR, AphA,Pex, CatM, AtzR, CatR, ClcR, CbbR, CysB, CbnR, OxyR, OccR, and CrgA.

In various embodiments, engineered protein sensor and/or switch is anengineered version of aN E. coli TF, such as ArcA, AtoC, BaeR, BasR,CitB, CpxR, CreB, CusR, DcuR, DpiA, EvgA, KdpE, NarL, NarP, OmpR, PhoB,

PhoP, QseB, RcsB, RstA, TorR, UhpA, UvrY, YedW, YehT, YfhK, YgiX, YpdB,ZraR, RssB, AgaR, AIIR (ybbU), ArsR, AscG, Betl, BgIJ, CadC, CaiF, CeiD,CueR, CynR, ExuR, FecR, FucR, Fur, GatR, GutM, GutR (SrIR), ModE, MtIR,NagC, NanR (yhcK), NhaR, PhnF, PutA, RbsR, RhaR, RhaS, RpiR (AlsR),SdiA, UidR, XapR, XyIR, ZntR, AlIS (ybbS), Arac, ArgR, AsnC, CysB, CytR,DsdC, GaIR, GaiS, GcvA, GcvR, GIcC, GlpR, GntR, IdnR, LctR, Lrp, LysR,MeiR, MhpR, TdcA, TdcR, TetR, TreR, TrpR, and TyrR.

In various embodiments, the engineered protein sensor and/or switch isan engineered version of a plant transcriptional regulator family suchas a member of the AP2, C2H2, Dof, LATA, HD-ZIP, M-type, NF-YA,S1Fa-like, TCP, YABBY, ARF, C3H, E2F/DP, GRAS, HRT-like, MIKC, NF-YB,SAP, Trihelix, ZF-HD, ARR-B, CAMTA, EIL, GRF, HSF, MYB, NF-YC, SBP, VOZ,bHLH, B3, CO-like, ERF, GeBP, LBD, MYB _related, NZZ/SPL, SRS, WOX,bZIP, BBR-BPC, CPP, FAR1, HB-PHD, LFY, NAC, Nin-like, STAT, WRKY, BES1,DBB, G2-like, HB-other, LSD, NF-X1, RAV, TALE, and Whirly families.

In various embodiments, the engineered protein sensor and/or switch isan engineered version of a yeast TF, such as Abf1p, Abf2p, Aca1p, Ace2p,Adr1p, Aft1p, Aft2p, Arg80p, Arg81p, Aro80p, Arr1p, Asg1p, Ash1p, Azf1p,Bas1p, Cad1p, Cat8p, Cbf1p, Cep3p, Cha4p, Cin5p, Crz1p, Cst6p, Cup2p,Cup9p, DaI80p, DaI81p, Dai82p, Dot6p, Ecm22p, Ecm23p, Eds1p, Ert1p,Fhl1p, Fkh1p, Fkh2p, Flo8p, Fzf1p, Gai4p, Gat1p, Gat3p, Gat4p, Gcn4p,Gcr1p, Gis1p, GIn3p, Gsm1p, Gzf3p, Haa1p, Hac1p, Hai9p, Hap1p, Hap2p,Hap3p, Hap4p, Hap5p,

Hcm1p, Hmlalpha2p, Hmra2p, Hsf1p, Ime1p, Ino2p, Ino4p, Ixr1p, Kar4p,Leu3p, Lys14p, Mac1p, Mai63p, Matalpha2p, Mbp1p, Mcm1p, Met31p, Met32p,Met4p, Mga1p, Mig1p, Mig2p, Mig3p, Mot2p, Mot3p, Msn1p, Msn2p, Msn4p,Mss11p, Ndt80p, Nhp10p, Nhp6ap, Nhp6bp, Nrg1p, Nrg2p, Oaf1p, Pdr1p,Pdr3p, Pdr8p, Phd1p, Pho2p, Pho4p, Pip2p, Ppr1p, Put3p, Rap1p, Rdr1p,Rds1p, Rds2p, Reb1p, Rei1p, Rfx1p, Rgm1p, Rgt1p, Rim101p, RIm1p, Rme1p,Rox1p, Rph1p, Rpn4p, Rsc30p, Rsc3p, Rsf2p, Rtg1p, Rtg3p, Sfl1p, Sfp1p,Sip4p, Skn7p, Sko1p, Smp1p, Sok2p, Spt15p, Srd1p, Stb3p, Stb4p, Stb5p,Ste12p, Stp1p, Stp2p, Stp3p, Stp4p, Sum1p, Sut1p, Sut2p, Swi4p, Swi5p,Tbf1p, Tbs1p, Tea1p, Tec1p, Tod6p, Tos8p, Tye7p, Uga3p, Ume6p, Upc2p,Urc2p, Usv1p, Vhr1p, War1p, Xbp1p, YER064C, YER130C, YER184C, YGRO67C,YKL222C, YLL054C, YLR278C, YML081W, YNR063W, YPR013C, YPR015C, YPR022C,YPR196W, Yap1p, Yap3p, Yap5p, Yap6p, Yap7p, Yox1p, Yrm1p, Yrr1p, andZap1p.

In various embodiments, the engineered protein sensor and/or switch isan engineered version of a nematode TF, such as ada-2, aha-1, ahr-1,alr-1, ast-1, atf-2, atf-5, atf-6, atf-7, athp-1, blmp-1, bra-2, brc-1,cbp-1, ccr-4, cdk-9, ced-6, ceh-1, ceh-10, ceh-12, ceh-13, ceh-14,ceh-16, ceh-17, ceh-18, ceh-19, ceh-2, ceh-20, ceh-21, ceh-22, ceh-23,ceh-24, ceh-26, ceh-27, ceh-28, ceh-30, ceh-31, ceh-32, ceh-33, ceh-34,ceh-36, ceh-37, ceh-38, ceh-39, ceh-40, ceh-41, ceh-43, ceh-44, ceh-45,ceh-48, ceh-49, ceh-5, ceh-6, ceh-60, ceh-7, ceh-8, ceh-9, cep-1, ces-1,ces-2, cey-1, cey-2, cey-3, cey-4, cfi-1, chd-3, cky-1, cnd-1, cog-1,crh-1, daf-12, daf-14, daf-16, daf-19, daf-3, daf-8, dcp-66, die-1,dlx-1, dmd-3, dmd-4, dmd-5, dmd-6, dnj-11, dpi-1, dpr-1, dpy-20, dpy-22,dpy-26, dro-1, dsc-1, efl-1, ef1-2, egl-13, egl-18, eg1-27, eg1-38,eg1-43, eg1-44, eg1-46, eg1-5, ek1-2, ek1-4, elc-1, elt-1, elt-2, elt-3,elt-4, elt-6, elt-7, end-1, end-3, eor-1, ets-4, ets-5, eya-1, fax-1,fkh-10, fkh-2, fkh-3, fkh-4, fkh-5, fkh-6, fkh-7, fkh-8, fkh-9, flt-1,fos-1, fozi-1, gei-11, gei-13, gei-3, gei-8, gfl-1, gla-3, ham-2, hbl-1,hif-1, hlh-1, hlh-10, hlh-11, hlh-12, hlh-13, hlh-14, hlh-15, hlh-16,hlh-17, hlh-19, hlh-2, hlh-25, hlh-26, hlh-27, hlh-28, hlh-29, hlh-3,hlh-30, hlh-4, hlh-6, hlh-8, hmg-1.1, hmg-1.2, hmg-1.2, hmg-11, hmg-12,hmg-3, hmg-4, hmg-5, hnd-1, hsf-1, irx-1, lag-1, let-381, let-418,Ifi-1, lim-4, lim-6, lim-7, lin-1, lin-11, lin-22, lin-26, lin-28,lin-31, lin-32, lin-35, lin-39, lin-40, lin-41, lin-48, lin-49, lin-54,lin-59, lin-61, hr-1, Ipd-2, Is1-1, Iss-4, Ist-3, mab-23, mab-3, mab-5,mab-9, mbf-1, mbr-1, mbr-1, mdl-1, mec-3, med-1, med-2, mef-2, mes-2,mes-4, mes-6, mex-1, mex-5, mex-6, mg1-2, mls-1, mis-2, mml-1, mua-1,mxl-1, mx1-2, mx1-3, nfi-1, ngn-1, nhr-1, nhr-10, nhr-100, nhr-101,nhr-102, nhr-103, nhr-104, nhr-105, nhr-106, nhr-107, nhr-108, nhr-109,nhr-11, nhr-110, nhr-111, nhr-112, nhr-113, nhr-114, nhr-115, nhr-116,nhr-117, nhr-118, nhr-119, nhr-12, nhr-120, nhr-121, nhr-122, nhr-123,nhr-124, nhr-125, nhr-126, nhr-127, nhr-128, nhr-129, nhr-13, nhr-130,nhr-131, nhr-132, nhr-133, nhr-134, nhr-135, nhr-136, nhr-137, nhr-138,nhr-139, nhr-14, nhr-140, nhr-141, nhr-142, nhr-143, nhr-145, nhr-146,nhr-147, nhr-148, nhr-149, nhr-15, nhr-150, nhr-152, nhr-153, nhr-154,nhr-155, nhr-156, nhr-157, nhr-158, nhr-159, nhr-16, nhr-161, nhr-162,nhr-163, nhr-164, nhr-165, nhr-166, nhr-167, nhr-168, nhr-169, nhr-17,nhr-170, nhr-171, nhr-172, nhr-173, nhr-174, nhr-175, nhr-176, nhr-177,nhr-178, nhr-179, nhr-18, nhr-180, nhr-181, nhr-182, nhr-183, nhr-184,nhr-185, nhr-186, nhr-187, nhr-188, nhr-189, nhr-19, nhr-190, nhr-191,nhr-192, nhr-193, nhr-194, nhr-195, nhr-196, nhr-197, nhr-198, nhr-199,nhr-2, nhr-20, nhr-201, nhr-202, nhr-203, nhr-204, nhr-205, nhr-206,nhr-207, nhr-208, nhr-209, nhr-21, nhr-210, nhr-211, nhr-212, nhr-213,nhr-214, nhr-215, nhr-216, nhr-217, nhr-218, nhr-219, nhr-22, nhr-220,nhr-221, nhr-222, nhr-223, nhr-225, nhr-226, nhr-227, nhr-228, nhr-229,nhr-23, nhr-230, nhr-231, nhr-232, nhr-233, nhr-234, nhr-237, nhr-238,nhr-239, nhr-241, nhr-242, nhr-243, nhr-244, nhr-245, nhr-246, nhr-247,nhr-248, nhr-249, nhr-25, nhr-250, nhr-251, nhr-252, nhr-253, nhr-254,nhr-255, nhr-256, nhr-257, nhr-258, nhr-26, nhr-260, nhr-261, nhr-262,nhr-263, nhr-264, nhr-265, nhr-266, nhr-267, nhr-268, nhr-269, nhr-27,nhr-270, nhr-271, nhr-272, nhr-273, nhr-274, nhr-275, nhr-276, nhr-277,nhr-278, nhr-28, nhr-280, nhr-281, nhr-282, nhr-283, nhr-285, nhr-286,nhr-288, nhr-3, nhr-30, nhr-31, nhr-32, nhr-33, nhr-34, nhr-35, nhr-36,nhr-37, nhr-38, nhr-39, nhr-4, nhr-40, nhr-41, nhr-42, nhr-43, nhr-44,nhr-45, nhr-46, nhr-47, nhr-47, nhr-48, nhr-49, nhr-5, nhr-50, nhr-51,nhr-52, nhr-53, nhr-54, nhr-55, nhr-56, nhr-57, nhr-58, nhr-59, nhr-6,nhr-60, nhr-61, nhr-62, nhr-63, nhr-64, nhr-65, nhr-66, nhr-67, nhr-68,nhr-69, nhr-7, nhr-70, nhr-71, nhr-72, nhr-73, nhr-74, nhr-75, nhr-76,nhr-77, nhr-78, nhr-79, nhr-8, nhr-80, nhr-81, nhr-82, nhr-83, nhr-84,nhr-85, nhr-86, nhr-87, nhr-88, nhr-89, nhr-9, nhr-90, nhr-91, nhr-92,nhr-94, nhr-95, nhr-96, nhr-97, nhr-98, nhr-99, nob-1, ntl-2, ntl-3,nurf-1, odr-7, oma-1, oma-2, pag-3, pal-1, pax-1, pax-3, peb-1, pes-1,pha-1, pha-2, pha-4, php-3, pie-1, pop-1, pos-1, pqn-47, pqn-75, psa-1,rabx-5, rbr-2, ref-1, mt-1, sbp-1, sdc-1, sdc-2, sdc-3, sea-1, sem-4,sex-1, skn-1, sknr-1, sma-2, sma-3, sma-4, smk-1, sop-2, sox-1, sox-2,sox-3, spr-1, sptf-2, sptf-3, srab-2, srt-58, srw-49, sta-1, tab-1,taf-4, taf-5, tag-153, tag-182, tag-185, tag-192, tag-295, tag-331,tag-347, tag-350, tag-68, tag-97, tbx-11, tbx-2, tbx-30, tbx-31, tbx-32,tbx-33, tbx-34, tbx-35, tbx-36, tbx-37, tbx-38, tbx-39, tbx-40, tbx-41,tbx-7, tbx-8, tbx-9, tra-1, tra-4, ttx-1, ttx-3, unc-120, unc-130,unc-3, unc-30, unc-37, unc-39, unc-4, unc-42, unc-55, unc-62, unc-86,vab-15, vab-3, vab-7, xbp-1, zag-1, zfp-1, zim-1, zip-1, zip-2, zip-3,zip-4, zip-5, and ztf-7.

In various embodiments, the engineered protein sensor and/or switch isan engineered version of a archeal TF, such as APE_0290.1, APE_0293,APE_0880b, APE_1602a, APE_2413, APE_2505, APE_0656a, APE_1799a,

APE_1458a, APE_1495a, APE_2570.1, APE_0416b.1, APE_0883a, APE_0535,APE_0142, APE_2021.1, APE_0060.1, APE_0197.1, APE_0778, APE_2011.1,APE_0168.1, APE_2517.1, APE_0288, APE_0002, APE_1360.1, APE_2091.1,APE_0454, APE1 862.1, APE_0669.1, APE_2443.1, APE_0787.1, APE_2004.1,APE_0025.1, APE_0153.1, AF0653, AF1264, AF1270, AF1544, AF1743, AF1807,AF1853, AF2008, AF2136, AF2404, AF0529, AF0114, AF0396, AF1298, AF1564,AF1697, AF1869, AF2271, AF1404, AF1148, AF0474, AF0584, AF1723, AF1622,AF1448, AF0439, AF1493, AF0337, AF0743, AF0365, AF1591, AF0128, AF0005,AF1745, AF0569, AF2106, AF1785, AF1984, AF2395, AF2232, AF0805, AF1429,AF0111, AF1627, AF1787, AF1793, AF1977, AF2118, AF2414, AF0643, AF1022,AF1121, AF2127, AF0139, AF0363, AF0998, AF1596, AF0673, AF2227, AF1542,AF2203, AF1459, AF1968, AF1516, AF0373, AF1817, AF1299, AF0757, AF0213,AF1009, AF1232, AF0026, AF1662, AF1846, AF2143, AF0674, Cmaq_0146,Cmaq_0924, Cmaq_1273, Cmaq_1369, Cmaq_1488, Cmaq_1508, Cmaq_1561,Cmaq_1699, Cmaq_0215, Cmaq_1704, Cmaq_1956, Cmaq_0058, Cmaq_1637,Cmaq_0227, Cmaq_0287, Cmaq_1606, Cmaq_1720, Cmaq_0112, Cmaq_1149,Cmaq_1687, Cmaq_0411, Cmaq_1925, Cmaq_0078, Cmaq_0314, Cmaq_0768,Cmaq_1206, Cmaq_0480, Cmaq_0797, Cmaq_1388, Cmaq_0152, Cmaq_0601,Cmaq_1188, Mboo_0375, Mboo_0423, Mboo_0749, Mboo_1012, Mboo_1134,Mboo_1154, Mboo_1189, Mboo_1266, Mboo_1711, Mboo_1971, Mboo_0002,Mboo_0956, Mboo_1071, Mboo_1405, Mboo_1643, Mboo_0973, Mboo_1170,Mboo_0158, Mboo_0195, Mboo_0277, Mboo_1462, Mboo_1574, Mboo_1649,Mboo_2112, Mboo_0013, Mboo_0386, Mboo_0946, Mboo_0977, Mboo_1081,Mboo_2241, Mboo_0142, Mboo_0396, Mboo_0409, Mboo_0976, Mboo_2244,Mboo_0526, Mboo_0346, Mboo_1018, Mboo_0917, Mboo_0323, Mboo_0916,Mboo_1680, Mboo_1288, Mboo_2311, Mboo_2048, Mboo_1027, Mboo_2312,rrnAC0161, rrnAC0578, rrnAC0961, rrnAC3494, rrnB0118, pNG7045, pNG6160,rrnAC0867, rrnAC2723, rrnAC3399, rrnAC3447, rrnB0052, rrnAC1653,rrnAC2779, pNG7038, rrnAC1252, rrnAC3288, rrnAC3307, rrnAC0503,rrnAC1269, pNG6047, rrnAC2622, rrnAC3290, rrnAC3365, rrnAC2301, pNG6157,rrnAC2002, rrnAC1238, rrnAC3207, pNG2039, pNG7160, rrnAC2748, rrnB0134,rrnAC2283, rrnAC1714, rrnAC1715, rrnAC2338, rrnAC2339, rrnAC2900,rrnAC0341, rrnAC3191, rrnAC1825, rrnAC2037, rrnAC0496, rrnAC3074,rrnAC2669, rrnAC0019, rrnACO231, rrnAC0564, rrnAC0640, rrnAC 1193, rrnAC1687, rrnAC 1786, rrnAC 1895, rrnAC1953, rrnAC 1996, rrnAC2017,rrnAC2022, rrnAC2052, rrnAC2070, rrnAC2160, rrnAC2472, rrnAC2785,rrnAC2936, rrnAC3167, rrnAC3451, rrnAC3486, rrnAC3490, rrnB0253,rrnB0269, pNG7159, pNG7188, pNG7357, pNG6134, rrnAC0376, rrnAC1217,rrnAC1541, rrnAC1663, rrnAC3229, pNG7223, rrnAC0440, rrnAC0535,rrnAC1742, rrnAC2519, rrnAC1764, rrnAC1777, rrnAC2762, rrnAC3264,rrnAC0417, rrnAC1303, rrnB0301, pNG6155, pNG7021, pNG7343, rrnAC1964,pNG7171, rrnAC1338, pNG7344, rrnACO230, rrnAC1971, rrnB0222, rrnAC0385,rrnAC0312, pNG7133, rrnAC0006, rrnAC1805, rrnAC3501, pNG7312, rrnAC0435,rrnAC0768, rrnAC0992, rrnAC2270, rrnAC3322, rrnB0112, rrnB0157,rrnB0161, pNG6058, pNG6092, pNG5119, pNG5140, pNG4042, pNG2006, pNG1015,rrnAC0199, rrnAC0681, rrnAC1765, rrnAC1767, pNG5067, pNG7180, pNG7307,pNG7183, rrnAC3384, pNG5131, rrnAC2777, pNG5071, rrnAC1472, pNG7308,rrnAC0869, rrnB0148, rrnAC2051, rrnAC0016, rrnAC1875, pNG6072, pNG6123,rrnAC2769, rrnAC1357, rrnAC1126, rrnAC0861, rrnAC0172, rrnAC0420,rrnAC0914, rrnAC2354, rrnAC3310, rrnAC3337, pNG5013, pNG5133, rrnAC3082,rrnB0074, pNG6075, pNG5024, rrnAC0924, rrnB0235, pNG7146, VNG0462C,VNG7122, VNG7125, VNG2445C, VNG0591C, VNG1843C, VNG0320H, VNG1123Gm,VNG1237C, VNG1285G, VNG2094G, VNG1351G, VNG1377G, VNG1179C, VNG1922G,VNG1816G, VNG0134G, VNG0194H, VNG0147C, VNG6193H, VNG2163H, VNG0101G,VNG1836G, VNG0530G, VNG0536G, VNG0835G, VNG2579G, VNG6349C, VNG1394H,VNG0113H, VNG0156C, VNG0160G, VNG0826C, VNG0852C, VNG1207C, VNG1488G,VNG6065G, VNG6461G, VNG7048, VNG7161, VNG1464G, VNG1548C, VNG0247C,VNG0471C, VNG0878Gm, VNG1029C, VNG1616C, VNG2112C, VNG6009H, VNG7007,VNG0704C, VNG1405C, VNG6318G, VNG0142C, VNG6072C, VNG6454C, VNG7053,VNG7156, VNG0703H, VNG0258H, VNG0751C, VNG1426H, VNG2020C, VNG6048H,VNG6126H, VNG6239G, VNG6478H, VNG7102, VNG6027G, VNG7023, VNG1786H,VNG2629G, VNG1598a, VNG7031, VNG6037G, VNG7171, VNG7114, VNG7038,VNG2243G, VNG6140G, VNG7100, VNG6476G, VNG6438G, VNG6050G, VNG0726C,VNG1390H, VNG6351G, VNG2184G, VNG0869G, VNG0254G, VNG6389G, VNG0315G,VNG0734G, VNG0757G, VNG1451C, VNG1886C, VNG1903Cm, VNG0985H, VNG6377H,HQ2607A, HQ2612A, HQ2779A, HQ1740A, HQ1541A, HQ1491A, HQ2619A, HQ1811A,HQ3063A, HQ3354A, HQ3642A, HQ2773A, HQ1436A, HQ2221A, HQ1414A, HQ3339A,HQ2484A, HQ3265A, HQ3620A, HQ1268A, HQ1388A, HQ1866A, HQ1563A, HQ1710A,HQ1962A, HQ1084A, HQ1739A, HQ1861A, HQ1863A, HQ2750A, HQ2664A, HQ2869A,HQ3058A, HQ3361A, HQ1277A, HQ2225A, HQ1993A, HQ1937A, HQ1088A, HQ1724A,HQ1568A, HQ2167A, HQ1230A, HQ2407A, HQ3108A, HQ1973A, HQ3260A, HQ2527A,HQ3410A, HQ2369A, HQ2564A, HQ1153A, HQ1227A, HQ3654A, HQ1867A, HQ2571A,HQ1625A, HQ3408A, HQ1689A, HQ2491A, HQ2726A, HQ2987A, HQ1041A, HQ1898A,HQ1900A, HQ1118A, Hbut_1261, Hbut_0073, Hbut_0009, Hbut_0100, Hbut_0987,Hbut_1340, Hbut_0120, Hbut_0990, Hbut_0316, Hbut_0659, Hbut_0660,Hbut_0366, Hbut_0204, Hbut_1 498, Hbut_1630, Hbut_1485, Hbut_1260,Hbut_0942, Hbut_0163, Hbut_0116, Hbut_0207, Hbut_1516, Hbut_0476,Hbut_1139, Hbut_0299, Hbut_0033, Hbut_0336, Hbut_1471, Hbut_1522,Hbut_0601, Hbut_0934, Hbut_0458, Hbut_0054, Hbut_1136, Hbut_0646,Hbut_0815, Igni_0122, Igni_0494, Igni_0706, Igni_1249, Igni_0226,Igni_0308, Igni_0658, Igni_0702, Igni_0486, Igni_0602, Igni_1394,Igni_0858, Igni_1361, Igni_0354, Igni_0989, Igni_1372, Igni_1124,Msed_0229, Msed_0717, Msed_1005, Msed_1190, Msed_1224, Msed_1970,Msed_2175, Msed_0166, Msed_0688, Msed_1202, Msed_1209, Msed_1765,Msed_1956, Msed_2295, Msed_0619, Msed_0621, Msed_2232, Msed_0140,Msed_2016, Msed_0767, Msed_1126, Msed_0856, Msed_0992, Msed_1773,Msed_1818, Msed_2183, Msed_1598, Msed_1725, Msed_2276, Msed_2293,Msed_1450, Msed_0265, Msed_0492, Msed_1279, Msed_1397, Msed_1563,Msed_1566, Msed_2027, Msed_0565, Msed_0868, Msed_1371, Msed_1483,Msed_1728, Msed_1351, Msed_1733, Msed_2209, Msed_2279, Msed_2233,MTH107, MTH517, MTH899, MTH1438, MTH1795, MTH163, MTH1288, MTH1349,MTH864, MTH1193, MTH254, MTH821, MTH1696, MTH739, MTH603, MTH214,MTH936, MTH659, MTH700, MTH729, MTH967, MTH1553, MTH1328, MTH1470,MTH1285, MTH1545, MTH931, MTH313, MTH1569, MTH281, MTH1488, MTH1521,MTH1627, MTH1063, MTH1787, MTH885, MTH1669, MTH1454, Msm_1107, Msm_1126,Msm_1350, Msm_1032, Msm_0213, Msm_0844, Msm_1260, Msm_0364, Msm_0218,Msm_0026, Msm_0329, Msm_0355, Msm_0453, Msm_1150, Msm_1408, Msm_0864,Msm_0413, Msm_1230, Msm_1499, Msm_1417, Msm_1250, Msm_1090, Msm_0720,Msm_0650, Msm_0424, Msm_0631, Msm_1445, Mbur_0656, Mbur_1148, Mbur_1658,Mbur_1965, Mbur_2405, Mbur_1168, Mbur_0166, Mbur_0946, Mbur_1817,Mbur_1830, Mbur_0231, Mbur_0234, Mbur_2100, Mbur_1375, Mbur_2041,Mbur_0776, Mbur_0783, Mbur_2071, Mbur_1477, Mbur_1871, Mbur_1635,Mbur_1221, Mbur_0292, Mbur_0512, Mbur_0609, Mbur_0661, Mbur_1211,Mbur_1719, Mbur_1811, Mbur_1931, Mbur_2112, Mbur_2130, Mbur_2048,Mbur_2144, Mbur_0368, Mbur_1483, Mbur_2274, Mbur_1359, Mbur_2306,Mbur_1647, Mbur_0631, Mbur_0378, Mbur_0085, Mbur_1496, Mbur_0963,Mbur_0372, Mbur_1140, Mbur_2097, Mbur_2262, Mbur_1532, Maeo_0092,Maeo_0872, Maeo_0888, Maeo_1298, Maeo_1146, Maeo_1061, Maeo_1147,Maeo_0865, Maeo_0659, Maeo_0679, Maeo_1305, Maeo_0977, Maeo_1182,Maeo_1472, Maeo_1362, Maeo_0019, Maeo_0277, Maeo_0356, Maeo_0719,Maeo_1032, Maeo_1289, Maeo_0698, Maeo_1183, Maeo_0223, Maeo_0822,Maeo_0218, Maeo_0186, Maeo_1155, Maeo_0575, Maeo_0728, Maeo_0696,Maeo_0664, MJ0432, MJ1082, MJ1325, MJ0229, MJ0361, MJ1553, MJ1563,MJ0774, MJ1398, MJ0723, MJ0151, MJ0589a, MJECL29, MJ1647, MJ1258,MJ0168, MJ0932, MJ0080, MJ0549, MJ0767, MJ1679, MJ0568, MJ1005, MJ0529,MJ0586, MJ0621, MJ1164, MJ1420, MJ1545, MJ0272, MJ0925, MJ0300, MJ1120,MJ0379, MJ0558, MJ1254, MJ0159, MJ0944, MJ0241, MJ0173, MJ0507, MJ0782,MJ0777, MJ1503, MJ1623, MmarC5_0244, MmarC5_1146, MmarC5_0136,MmarC5_1648, MmarC5_1124, MmarC5_0967, MmarC5_1647, MmarC5_0448,MmarC5_0231, MmarC5_0579, MmarC5_1252, MmarC5_1664, MmarC5_0974,MmarC5_0625, MmarC5_1666, MmarC5_0111, MmarC5_1039, MmarC5_0316,MmarC5_0131, MmarC5_1762, MmarC5_1579, MmarC5_0380, MmarC5_0898,MmarC5_0813, MmarC5_1143, MmarC5_1694, MmarC5_1294, MmarC5_1236,MmarC5_1150, MmarC5_1138, MmarC5_1543, MmarC5_0999, MmarC5_1507,MmarC5_0876, MmarC5_0202, MmarC5_1416, MmarC5_0612, MmarC5_0571,MmarC5_1100, MmarC5_1639, MmarC5_1644, MmarC5_0714, MmarC5_0484,MmarC5_0976, MmarC6_0024, MmarC6_0026, MmarC6_0104, MmarC6_0105,MmarC6_0128, MmarC6_0252, MmarC6_0566, MmarC6_0917, MmarC6_1231,MmarC6_0916, MmarC6_1531, MmarC6_0524, MmarC6_1326, MmarC6_1644,MmarC6_0165, MmarC6_0929, MmarC6_0258, MmarC6_0037, MmarC6_0055,MmarC6_1206, MmarC6_1606, MmarC6_0210, MmarC6_0325, MmarC6_0744,MmarC6_0850, MmarC6_1025, MmarC6_1226, MmarC6_1398, MmarC6_1462,MmarC6_1664, MmarC6_1175, MmarC6_0959, MmarC6_0931, MmarC6_0136,MmarC6_0425, MmarC6_0508, MmarC6_0285, MmarC6_0184, MmarC6_0443,MmarC6_0782, MmarC6_1297, MmarC6_0861, MmarC6_0696, MmarC6_1636,MmarC6_1817, MmarC6_0908, MmarC6_0913, MmarC6_0262, MmarC6_1567,MmarC6_1748, MmarC7_0274, MmarC7_0687, MmarC7_1029, MmarC7_1513,MmarC7_1661, MmarC7_1030, MmarC7_0388, MmarC7_0257, MmarC7_0592,MmarC7_1384, MmarC7_1017, MmarC7_1655, MmarC7_0306, MmarC7_0712,MmarC7_0235, MmarC7_0457, MmarC7_0521, MmarC7_0692, MmarC7_0743,MmarC7_0919, MmarC7_1096, MmarC7_1211, MmarC7_1587, MmarC7_1702,MmarC7_0987, MmarC7_1015, MmarC7_0031, MmarC7_1400, MmarC7_1790,MmarC7_1499, MmarC7_1629, MmarC7_1168, MmarC7_1727, MmarC7_0621,MmarC7_1085, MmarC7_1260, MmarC7_0085, MmarC7_0265, MmarC7_1461,MmarC7_1038, MmarC7_1033, MmarC7_0154, MmarC7_0352, MmarC7_1652,MmarC7_1455, MMP0499, MMP1442, MMP0480, MMP0752, MMP0032, MMP0460,MMP0637, MMP0033, MMP0217, MMP1137, MMP0386, MMP1347, MMP1015, MMP0719,MMP0020, MMP0631, MMP0742, MMP1467, MMP1052, MMP0097, MMP0209, MMP0568,MMP0674, MMP0678, MMP0993, MMP1210, MMP1275, MMP1447, MMP1646, MMP1499,MMP0018, MMP1712, MMP0402, MMP0787, MMP0607, MMP0168, MMP0700, MMP0465,MMP1376, MMP0086, MMP0257, MMP0840, MMP1023, MMP0791, MMP0799, MMP0041,MMP0036, MMP0907, MMP0629, MMP1100, Mevan_0753, Mevan_1029, Mevan_1232,Mevan_1560, Mevan_1502, Mevan_1030, Mevan_0459, Mevan_0343, Mevan_0658,Mevan_1373, Mevan_1201, Mevan_1594, Mevan_1567, Mevan_1203, Mevan_0375,Mevan_0778, Mevan_0320, Mevan_0525, Mevan_0587, Mevan_0758, Mevan_0808,Mevan_0951, Mevan_1109, Mevan_1444, Mevan_1514, Mevan_1517, Mevan_1014,Mevan_0136, Mevan_0295, Mevan_1389, Mevan_1479, Mevan_1173, Mevan_1578,Mevan_1653, Mevan_0686, Mevan_1098, Mevan_1270, Mevan_0270, Mevan_0282,Mevan_1620, Mevan_1668, Mevan_1038, Mevan_1044, Mevan_1050, Mayan _1056,Mayan _1033, Mevan_0014, Mevan_0425, Mevan_0095, Mlab_0303, Mlab_0817,Mlab_0821, Mlab_1236, Mlab_1381, Mlab_0824, Mlab_0002, Mlab_0494,Mlab_0162, Mlab_0744, Mlab_1629, Mlab_0854, Mlab_0909, Mlab_1549,Mlab_0037, Mlab_0071, Mlab_0160, Mlab_1173, Mlab_1603, Mlab_1630,Mlab_1666, Mlab_1628, Mlab_0070, Mlab_1522, Mlab_0331, Mlab_1259,Mlab_0324, Mlab_1366, Mlab_1576, Mlab_0353, Mlab_0010, Mlab_0295,Mlab_0588, Mlab_1668, Mlab_0447, Mlab_0440, Mlab_0197, Mlab_1697,Mlab_1694, Mlab_1710, Mlab_1511, Mlab_0458, Mlab_0497, Mlab_0762,Mlab_0988, Mlab_0826, Memar_0011, Memar_0013, Memar_1330, Memar_1512,Memar_1567, Memar_1770, Memar_2080, Memar_0129, Memar_0140, Memar_0431,Memar_1231, Memar_1756, Memar_2162, Memar_2068, Memar_1225, Memar_0002,Memar_1921, Memar_0834, Memar_2239, Memar_1448, Memar_0817, Memar_2411,Memar_2490, Memar_2264, Memar_1471, Memar_1420, Memar_0458, Memar_1291,Memar_1391, Memar_1410, Memar_1819, Memar_2218, Memar_2347, Memar_2360,Memar_2449, Memar_1304, Memar_0106, Memar_0096, Memar_0419, Memar_1120,Memar_0385, Memar_0555, Memar_1103, Memar_1319, Memar_2487, Memar_1252,Memar_1388, Memar_0473, Memar_1524, Memar_0459, Memar_0487, Memar_1209,Memar_1387, Memar_2116, MK0576, MK1025, MK0542, MK1515, MK0506, MK1677,MK1502, MK1190, MK0175, MK0800, MK0457, MK0449, MK1380, MK1430, MK0574,MK1482, MK0984, MK0337, MK1587, MK0839, MK0619, MK0858, MK0495, MK0253,Mthe_1108, Mthe_1291, Mthe_1230, Mthe_0612, Mthe_0503, Mthe_0879,Mthe_0047, Mthe_0598, Mthe_0023, Mthe_0662, Mthe_0543, Mthe_0154,Mthe_0459, Mthe_1389, Mthe_1446, Mthe_1633, Mthe_1233, Mthe_0669,Mthe_0067, Mthe_0404, Mthe_0982, Mthe_1201, Mthe_0152, Mthe_0265,Mthe_1650, Mthe_1683, Mthe_0889, MA0191, MA0342, MA0380, MA1458, MA2551,MA3784, MA3925, MA3940, MA3952, MA4076, MA4344, MA4484, MA4576, MA0207,MA0750, MA2499, MA3597, MA4479, MA2544, MA4480, MA0504, MA2921, MA0862,MA0205, MA0460, MA0622, MA0629, MA1953, MA4398, MA4560, MA0723, MA1529,MA1551, MA2421, MA1531, MA0924, MA0575, MA1588, MA0672, MA1395, MA4075,MA1763, MA2814, MA3468, MA0022, MA4338, MA2133, MA0971, MA1005, MA0067,MA1424, MA1815, MA4668, MA2914, MA3524, MA4040, MA4267, MA3984, MA0283,MA0333, MA0414, MA1339, MA3166, MA0176, MA0180, MA0743, MA1863, MA2051,MA2055, MA2206, MA2211, MA2771, MA3189, MA4167, MA1122, MA3015, MA0079,MA0989, MA4404, MA2093, MA1671, MA4106, MA4346, MA0278, MA4331, MA0179,MA2948, MA3586, MA2761, MA1487, MA1771, MA2746, MA0364, MA2951, MA0354,MA2902, MA0368, MA2764, MA2766, MA0178, MA2782, MA2493, MA0610, MA3871,MA0287, MA0359, MA1835, MA2057, MA2207, MA2212, MA3151, MA4622, MA0926,MA1664, MA4408, MA1868, Mbar_A0506, Mbar_A0581, Mbar_A0738, Mbar_A0909,Mbar_A1363, Mbar_A1705, Mbar_A1707, Mbar_A1708, Mbar_A1719, Mbar_A2323,Mbar_A2748, Mbar_A3221, Mbar_A3427, Mbar_A1541, Mbar_A1729, Mbar_A2416,Mbar_A3312, Mbar_A0803, Mbar_A3558, Mbar_A0794, Mbar_A2965, Mbar_A1070,Mbar_A1333, Mbar_A2865, Mbar_A1639, Mbar_A3371, Mbar_A0650, Mbar_A3377,Mbar_A3361, Mbar_A0654, Mbar_A3464, Mbar_A1460, Mbar_A2808, Mbar_A1584,Mbar_A2743, Mbar_A2250, Mbar_A0507, Mbar_A0992, Mbar_A1457, Mbar_A0588,Mbar_A0122, Mbar_A2068, Mbar_A0552, Mbar_A0621, Mbar_A0692, Mbar_A1033,Mbar_A2079, Mbar_A2171, Mbar_A2318, Mbar_A2819, Mbar_A2992, Mbar_A3339,Mbar_A1265, Mbar_A1377, Mbar_A1884, Mbar_A2294, Mbar_A3663, Mbar_A2575,Mbar_A2637, Mbar_A3146, Mbar_A3330, Mbar_A3493, Mbar_A2012, Mbar_A2036,Mbar_A2688, Mbar_A3560, Mbar_A1076, Mbar_A0340, Mbar_A0520, Mbar_A1497,Mbar_A3486, Mbar_A1949, Mbar_A0475, Mbar_A0579, Mbar_A1062, Mbar_A0595,Mbar_A3297, Mbar_A3442, Mbar_A3419, Mbar_A0834, Mbar_A0787, Mbar_A2740,Mbar_A1394, Mbar_A0196, Mbar_A1270, Mbar_A3331, Mbar_A3578, Mbar_A3670,Mbar_A1080, MM0272, MM0662, MM0841, MM1040, MM1257, MM1484, MM1796,MM2237, MM2242, MM2246, MM2247, MM2261, MM2525, MM2985, MM3068, MM3208,MM1882, MM1494, MM3092, MM1595, MM3173, MM0565, MM1492, MM0266, MM1080,MM1605, MM1650, MM2809, MM2861, MM2446, MM2441, MM2040, MM1728, MM1739,MM2416, MM1825, MM0666, MM0842, MM2657, MM1332, MM2573, MM1034, MM2606,MM0247, MM0444, MM0872, MM0927, MM1363, MM2394, MM2895, MM3179, MM1005,MM3233, MM1550, MM0359, MM0361, MM1586, MM1863, MM2851, MM2853, MM3117,MM0116, MM0289, MM0346, MM1903, MM3195, MM3170, MM1085, MM0386, MM2835,MM0811, MM1042, MM1027, MM2184, MM1028, MM0432, MM2546, MM1614, MM1772,MM0692, MM0146, MM0345, MM0369, MM1554, MM2854, MM1094, MM2042, MM3115,Msp_0061, Msp_0120, Msp_1519, Msp_0293, Msp_1556, Msp_0769, Msp_0168,Msp_0614, Msp_0518, Msp_0122, Msp_0383, Msp_1218, Msp_0446, Msp_0265,Msp_0608, Msp_1143, Msp_1207, Msp_0248, Msp_0512, Msp_0823, Msp_1188,Msp_0235, Msp_0194, Msp_1057, Msp_1097, Msp_0717, Msp_0971, Msp_1360,Msp_1272, Msp_1125, Msp_0149, Mhun_0040, Mhun_0316, Mhun_0873,Mhun_1073, Mhun_1644, Mhun_2448, Mhun_2633, Mhun_2472, Mhun_0365,Mhun_0919, Mhun_0576, Mhun_0165, Mhun_2458, Mhun_0842, Mhun_0941,Mhun_1324, Mhun_1346, Mhun_2089, Mhun_1313, Mhun_1731, Mhun_1706,Mhun_0152, Mhun_0501, Mhun_1037, Mhun_2548, Mhun_2928, Mhun_3036,Mhun_0241, Mhun_1541, Mhun_2190, Mhun_0646, Mhun_1347, Mhun_1533,Mhun_1553, Mhun_1866, Mhun_1954, Mhun_0253, Mhun_1259, Mhun_1451,Mhun_2502, Mhun_0684, Mhun_2259, Mhun_0763, Mhun_1327, Mhun_1530,Mhun_2935, Mhun_2804, Mhun_0568, Mhun_0593, Mhun_1236, Mhun_1656,Mhun_2481, Mhun_2797, Mhun_0497, Mhun_0575, Mhun_0588, NEQ328, NEQ229,NEQ348, NEQ288, NEQ453, NEQ143, NEQ039, NEQ276, NEQ098, NEQ541, NP1838A,NP2534A, NP3936A, NP6056A, NP2558A, NP1144A, NP0458A, NP2490A, NP2664A,NP3370A, NP0078A, NP5052A, NP4026A, NP6200A, NP0924A, NP4828A, NP2752A,NP6106A, NP2470A, NP2474A, NP0316A, NP0252A, NP5326A, NP1048A, NP2958A,NP5152A, NP4632A, NP3636A, NP3734A, NP4552A, NP5064A, NP1496A, NP4726A,NP2878A, NP0136A, NP0162A, NP0654A, NP1532A, NP1538A, NP1564A, NP2794A,NP4286A, NP4406A, NP5130A, NP5298A, NP6030A, NP6220A, NP4436A, NP1320A,NP2146A, NP3466A, NP4796A, NP5168A, NP3046A, NP2812A, NP3608A, NP2618A,NP6176A, NP3330A, NP7054A, NP2762A, NP4124A, NP3490A, NP1128A, NP1628A,NP2114A, NP0674A, NP2366A, NP3002A, NP3776A, NP4444A, NP1296A, NP1064A,NP4080A, NP4082A, NP0534A, NP2466A, NP3718A, NP5096A, NP2220A, NP5186A,NP1684A, NP2246A, NP4822A, NP4326A, NP4106A, NP2518A, NP5272A, NP6088A,NP4258A, PT00082, PT00457, PT00754, PT00795, PT00420, PT01287, PT00595,PT00891, PT00200, PT01201, PT00428, PT00376, PT00514, PT00375, PT00781,PT01148, PT00979, PT00276, PT00843, PT00557, PT01105, PT01211, PT01517,PT01052, PT01150, PT00114, PT01041, PT01176, PT00063, PT00799, PT01388,PT01389, PT00914, PT01110, PT01216, PT00675, PT01123, PT00506, PT01258,PT01372, PT00363, PT01340, PT01338, PT01067, PT01454, PT01523, PT00576,PT00198, PAE0731, PAE0738, PAE1612, PAE2042, PAE2911, PAE1948, PAE2655,PAE0385, PAE2225, PAE3116, PAE2186, PAE1103, PAE1592, PAE1848, PAE3387,PAE1507, PAE1986, PAE3469, PAE3471, PAE0659, PAE1443, PAE1484, PAE0296,PAE2022, PAE2357, PAE1544, PAE0640, PAE2309, PAE3163, PAE2449, PAE3605,PAE0783, PAE1627, PAE1638, PAE2071, PAE3208, PAE0019, PAE0813, PAE3327,PAE0146, PAE2679, PAE2684, PAE1218, PAE1760, PAE0013, PAE3437, PAE2640,PAE3378, PAE2164, PAE0171, PAE0170, PAE3329, PAE2120, PAE1645, PAE0781,PAE2282, Pars_0006, Pars_0433, Pars_0703, Pars_0836, Pars_0990,Pars_1924, Pars_2088, Pars_2298, Pars_0264, Pars_2028, Pars_0627,Pars_1855, Pars_2059, Pars_1853, Pars_0399, Pars_0425, Pars_1561,Pars_2084, Pars_0343, Pars_0668, Pars_2155, Pars_0438, Pars_1526,Pars_2364, Pars_1428, Pars_0037, Pars_1981, Pars_1988, Pars_2104,Pars_0057, Pars_0792, Pars_0504, Pars_0550, Pars_1742, Pars_1776,Pars_0311, Pars_0752, Pars_1087, Pars_1872, Pars_1005, Pars_0806,Pars_2186, Pars_2187, Pars_1743, Pars_2132, Pars_1649, Pars_1976,Pars_0035, Pars_1810, Pars_2125, Pcal_0142, Pcal_0905, Pcal_0946,Pcal_0412, Pcal_0495, Pcal_0687, Pcal_1273, Pcal_0822, Pcal_1595,Pcal_1185, Pcal_0610, Pcal_1183, Pcal_2085, Pcal_0796, Pcal_0536,Pcal_1689, Pcal_0008, Pcal_1198, Pcal_1653, Pcal_0295, Pcal_1924,Pcal_1927, Pcal_0200, Pcal_0589, Pcal_0596, Pcal_2145, Pcal_0791,Pcal_0023, Pcal_1415, Pcal_1735, Pcal_0266, Pcal_0346, Pcal_0543,Pcal_0792, Pcal_1032, Pcal_0159, Pcal_1078, Pcal_1890, Pcal_1316,Pcal_1055, Pcal_0584, Pcal_1734, Pcal_2147, Pcal_1638, Pcal_2070,Pisl_1759, Pisl_2001, Pisl_0858, Pisl_1838, Pisl_0307, Pisl_0653,Pisl_1426, Pisl_1248, Pisl_1639, Pisl_1808, Pisl_0995, Pisl_1590,Pisl_0997, Pisl_0709, Pisl_1563, Pisl_1834, Pisl_1578, Pisl_0622,Pisl_1613, Pisl_0725, Pisl_1023, Pisl_0410, Pisl_1076, Pisl_1655,Pisl_1662, Pisl_1854, Pisl_0045, Pisl_1100, Pisl_0810, Pisl_0572,Pisl_1971, Pisl_1303, Pisl_1717, Pisl_0038, Pisl_0979, Pisl_0565,Pisl_1878, Pisl_0807, Pisl_1975, Pisl_1974, Pisl_0573, Pisl_0955,Pisl_1667, Pisl_1074, Pisl_1008, Pisl_1250, PAB2298, PAB1869, PAB0625,PAB0751, PAB1002, PAB2328, PAB0125, PAB0208, PAB0619, PAB1229, PAB1227,PAB0108, PAB0322, PAB0392, PAB2312, PAB7115, PAB2062.1n, PAB1938,PAB1236, PAB2257, PAB7359, PAB2299, PAB0758a, PAB3089, PAB3117, PAB0960,PAB1522.1n, PAB2324, PAB0714, PAB2311, PAB1533, PAB0211, PAB2104,PAB2035, PAB0475, PAB0842, PAB0668, PAB7155, PAB3293, PAB0917, PAB0661,PAB0953, PAB1243, PAB1544, PAB0331, PAB1922, PAB7338, PAB0603, PAB1517,PAB1726, PAB1641, PAB1642, PAB0976, PAB1912, PAB0950, PAB0838, PF0007,PF0230, PF1072, PF1406, PF2051, PF0113, PF0232, PF1790, PF1088, PF0095,PF1734, PF0054, PF1543, PF1732, PF0250, PF0739, PF1231, PF1601, PF1022,PF1893, PF0607, PF0829, PF1722, PF1831, PF0322, PF0524, PF2053, PF0851,PF1194, PF0055, PF0505, PF0512, PF1386, PF1735, PF1794, PF1851, PF0691,PF0487, PF0988, PF1029, PF2062, PF0263, PF0709, PF1476, PF0584, PF1198,PF0535, PF1295, PF1338, PF1337, PF0687, PF1377, PF0491, PF0496, PF0661,PF1743, PF0124, PF0649, PH0062, PH1101, PH0199, PH0289, PH0825, PH1061,PH1406, PH1744, PH1930, PH1932, PH0977, PH0952, PH0180, PH1692, PH0045,PH1856.1n, PH0061, PHS045, PH1592, PH1916, PH0140, PH1519, PHS023,PH1055, PHS034, PHS051, PHSO46, PH0601, PHS024, PH0468, PH1163, PH0046,PH0787, PH0783, PH1471, PH1691, PH1748, PH1808, PH0660, PH0804, PH0995,PH0614, PH0914, PH0718.1n, PH1080, PH0763, PH1009, PH1161, PH1160,PH1482, PH0864, PH0619, PH0751, PH0799, PH1034, PH0588, Smar_0567,Smar_0017, Smar_0429, Smar_1295, Smar_0048, Smar_0184, Smar_0954,Smar_1451, Smar_0205, Smar_0336, Smar_0366, Smar_1141, Smar_0476,Smar_0879, Smar_0338, Smar_0194, Smar_0612, Smar_0915, Smar_1254,Smar_1341, Smar_0279, Smar_1409, Smar_0319, Smar_0758, Smar_1442,Smar_1514, Smar_1075, Smar_1322, Smar_0054, Smar_1137, Smar_1250,Smar_0918, Smar_0086, Saci_0006, Saci_0446, Saci_1068, Saci_1787,Saci_1979, Saci_0800, Saci_1710, Saci_2236, Saci_2266, Saci_2136,Saci_0992, Saci_0731, Saci_0752, Saci_1304, Saci_1588, Saci_0944,Saci_0843, Saci_0942, Saci_0264, Saci_1391, Saci_0476, Saci_1223,Saci_0112, Saci_0048, Saci_1851, Saci_0455, Saci_2061, Saci_2116,Saci_2167, Saci_2183, Saci_2296, Saci_0655, Saci_1344, Saci_1505,Saci_2359, Saci_1192, Saci_2313, Saci_0161, Saci_0102, Saci_0133,Saci_0874, Saci_1219, Saci_1482, Saci_1670, Saci_1956, Saci_2112,Saci_0488, Saci_0483, Saci_1180, Saci_1171, Saci_1186, Saci_1242,Saci_0489, Saci_1005, Saci_2352, Saci_0380, Saci_1336, Saci_1230,Saci_2283, Saci_1107, Saci_0866, Saci_1341, Saci_0652, Saci_0842,Saci_1161, SSO0458, SSO0620, SSO9953, SSO2688, SSO0200, SSO1423,SSO2114, SSO2347, SSO3103, SSO5522, SSO0977, SSO0606, SSO2131, SSO10340,SSO0157, SSO6024, SSO0659, SSO5826, SSO10342, SSO3242, SSO0669, SSO2273,SSO2244, SSO1589, SSO1255, SSO0447, SSO0785, SSO1008, SSO1219, SSO1306,SSO1536, SSO2058, SSO3061, SSO3080, SSO1868, SSO3097, SSO2474, SSO3188,SSO0107, SSO0270, SSO0387, SSO0942, SSO1066, SSO0040, SSO1264, SSO1384,SSO1750, SSO1897, SSO2090, SSO2132, SSO2933, SSO2992, SSO2897, SSO3176,SSO0048, SSO0365, SSO1082, SSO1108, SSO1352, SSO1101, SSO1110, SSO2652,SSO1695, SSO1748, SSO2957, SSO2327, SSO0038, SSO0049, SSO0994, SSO2138,SSO2571, SSO0951, SSO2206, SSO2089, SSO2598, SSO2506, SSO0446, SSO0946,SSO0266, SSO0426, SSO2073, STO236, ST1060, ST1064, ST1076, ST1486,ST1604, ST1889, STS229, STO720, STO173, STS095, ST2514, ST1022, ST2372,STO193, STO489, ST1115, ST1301, STSO42, ST1473, STS071, STS074, STS163,STS072, STS250, STS248, ST2039, ST2236, ST2114, ST2562, STO051, STO164,STO722, ST2550, ST1593, STO256, STO331, ST1268, ST2084, ST2190, ST1409,STO808, STS035, STO758, ST1043, ST1386, ST1710, ST1716, ST1867, ST1890,ST2388, STS086, STO749, STO837, STO980, ST2050, STO757, STO766, ST2210,ST1773, ST1340, ST1054, ST1275, ST1007, ST1041, STO684, STO072, STO349,ST1271, STO334, ST1630, STO371, TK0063, TK0559, TK1041, TK1261, TK1826,TK1881, TK2190, TK1086, TK1883, TK1955, TK2291, TK2134, TK1285, TK1487,TK0168, TK1331, TK0567, TK0834, TK1491, TK1210, TK2110, TK2052, TK0143,TK1413, TK2289, TK2270, TK1815, TK1439, TK0695, TK1259, TK0107, TK0448,TK1057, TK1058, TK1272, TK0697, TK0126, TK0539, TK1266, TK1688, TK2197,TK2218, TK1489, TK1339, TK0142, TK0169, TK1246, TK0770, TK1494, TK1924,TK2107, TK1143, TK1654, TK0151, TK0779, TK2151, TK0132, TK2287, TK1280,TK2024, TK0471, TK1769, TK1913, TK1050, Tpen_0466, Tpen_0552, Tpen_0860,Tpen_1509, Tpen_0232, Tpen_0836, Tpen_1499, Tpen_0577, Tpen_0018,Tpen_0579, Tpen_0150, Tpen_0366, Tpen_0869, Tpen_0668, Tpen_0348,Tpen_1236, Tpen_0124, Tpen_0102, Tpen_0973, Tpen_1621, Tpen_0378,Tpen_0538, Tpen_0707, Tpen_0776, Tpen_0069, Tpen_0090, Tpen_0173,Tpen_1796, Tpen_1358, Tpen_0115, Tpen_1464, Tpen_1595, Tpen_1401,Tpen_0901, Tpen_1818, Tpen_0293, Tpen_0690, Tpen_0374, Tpen_0710,Tpen_0070, Tpen_1551, Tpen_1591, Tpen_1154, Tpen_1562, Ta0472, Ta0731,Ta1110, Ta0115, Ta1173, Ta1443, Ta0185, Ta0678, Ta0608, Ta0257, Ta0981,Ta0093, Ta0550m, Ta0842, Ta0872, Ta1362m, Ta0736, Ta1394, Ta0166,Ta0675, Ta0748, Ta1231, Ta1186, Ta0106, Ta0948, Ta1282m, Ta1363, Ta0131,Ta0320m, Ta0411, Ta1064, Ta1166, Ta1218, Ta1503, Ta0201, Ta0346, Ta1496,Ta0868m, Ta1061m, Ta0825, Ta0795, Ta0199, Ta1485, Ta0945, Ta0940,Ta0134, Ta0685, Ta0890, Ta1324, TVN0192, TVN0983, TVN1251, TVN0658,TVN0295, TVN1196, TVN1337, TVN1127, TVN0160, TVN0945, TVN0938, TVN0292,TVN0236, TVN0364, TVN0447, TVN0906, TVN1422, TVN0185, TVN0291, TVN0514,TVN 1093, TVN0210, TVN 1272, TVN0519, TVN0603, TVN 1246, TVN 1408, TVN1203, TVN1162, TVN0516, TVN1265, TVN1392, TVN1493, TVN0934, TVN0728,TVN0704, TVN1394, TVN0084, TVN1083, TVN1089, TVN0213, TVN1149, TVN0972,TVN0377, LRC567, RCIX1274, RCIX1420, RCIX1655, RCIX1698, RCIX2213,RCIX2336, RRC298, RRC486, RRC76, RCIX1140, RCIX2193, RCIX670, RCIX684,RCIX808, RCIX820, LRC582, RCIX785, LRC109, RCIX103, RCIX105, RCIX106,RCIX1508, RCIX1739, RCIX2247, RRC465, RCIX1740, RCIX2328, RRC178,LRC575, RCIX1349, RCIX1520, LRC520, RCIX125, RCIX1430, RCIX148,RCIX1527, RCIX1743, RCIX2456, RCIX449, RCIX571, RRC212, RCIX960, LRC190,RCIX1230, RCIX414, RCIX1747, LRC319, RCIX1292, RCIX1376, RCIX2173,RCIX2196, RRC154, RCIX1238, RCIX1068, RCIX1190, RCIX1914, RCIX2177,RCIX824, RCIX989, RCIX2108, LRC274, LRC304, RCIX1189, RCIX1785,RCIX1790, and RCIX90.

In various embodiments, the engineered protein sensor and/or switch isan engineered version of a B. subtilis TF, such as Abh, AbrB, AcoR,AdaA, AhrC, AlaR, AIsR, AnsR, AraR, ArfM, ArsR, AzIB, BirA, BkdR, BItR,BmrR, CcpA, CcpB, CcpC, CggR, CheB, CheV, CheY, CitR, CitT, CodY, ComA,ComK, ComZ, CssR, CtsR, DctR, DegA, DegU, DeoR, DnaA, ExuR, FNR, FruR,Fur, GabR, GerE, GIcK, GIcR, GIcT, GInR, GIpP, GItC, GItR, GntR, GutR,Hbs, Hpr, HrcA, HtrA, HutP, HxIR, loiR, Ipi, KdgR, KipR, LacR, LevR,LexA, LicR, LicT, LmrA, LrpA, LrpB, LrpC, LytR, LytT, ManR, MecA, Med,MntR, MsmR, Mta, MtIR, MtrB, NhaX, PadR, PaiA, PaiB, PerR, Phage PBSXtranscriptional regulator, PhoP, PksA, PucR, PurR, PyrR, RbsR, ResD,Rho, RocR, Rok, RpIT, RsfA, SacT, SacV, SacY, SenS, SigA, SigB, SigD,SigE, SigF, SigG, SigH, Sigl, SigK, SigL, SigM, SigV, SigW, SigX, SigY,SigZ, SinR, Slr, SpIA, SpoOA, SpoOF, SpoIIID, SpoVT, TenA, Tenl, TnrA,TreR, TrnB-GIy1, TrnB-Phe, TrnD-Cys, TrnD-Gly, TrnD-Phe, TrnD-Ser,TrnD-Trp, TrnD-Tyr, Trnl-Gly, Trnl-Thr, TrnJ-Gly, TrnS-Leu2, TrnSL-Tyr1,TrnSL-VaI2, Xpf, Xre, XyIR, YacF, YazB, YbaL, YbbB, YbbH, YbdJ, YbfA,Ybfl, YbfP, YbgA, YcbA, YcbB, YcbG, YcbL, YccF, YccH, YceK, YcgE, YcgK,YcIA, YcIJ, YcnC, YcnK, YcxD, YczG, YdcH, YdcN, YdeB, YdeC, YdeE, YdeF,YdeL, YdeP, YdeS, YdeT, YdfD, YdfF, Ydfl, YdfL, YdgC, YdgG, YdgJ, YdhC,YdhQ, YdhR, YdiH, YdzF, YerO, YesN, YesS, YetL, YezC, YezE, YfhP, YfiA,YfiF, YfiK, YfiR, YfiV, YfmP, Yhbl, YhcB, YhcF, YhcZ, YhdE, Yhdl, YhdQ,YhgD, YhjH, YhjM, YisR, YisV, YjbD, Yjdl, YkmA, YkoG, YkoM, YkvE, YkvN,YkvZ, YlaC, YlbO, YIpC, YmfC, Ynel, YoaU, YobD, YobQ, YocG, YodB, YofA,YonR, YopO, YopS, YozA, YozG, YpbH, YpIP, YpoP, YpuH, YqaE, YqaF, YgaG,YqfL, YqzB, YraB, YraN, YrdQ, Yrhl, YrhM, YrkP, YrxA, YrzC, YsiA, YsmB,YtcD, YtdP, YtII, YtrA, YtsA, YttP, YtzE, YufM, YuIB, YurK, YusO, YusT,YuxN, YvaF, YvaN, YvaO, YvaP, YvbA, YvbU, YvcP, YvdE, YvdT, Yvfl, YyfU,YvhJ, YvkB, YvmB, YvnA, YvoA, YvqC, YvrH, Yvrl, YvyD, YvzC, YwaE, Ywbl,YwcC, YwfK, YwgB, YwhA, YwoH, YwqM, YwrC, YwtF, YxaD, YxaF, YxbF, YxdJ,YxjL, YxjO, YyaN, YybA, YybE, YybR, YycF, YydK, and Zur.

In various embodiments, the engineered protein sensor and/or switch isan engineered version of a Arabidopsis thaliana TF, such as AT1G01060,AT1G01380, AT1G01530, AT1G02340, AT1G04370, AT1G06160, AT1G07640,AT1G09530, AT1G09770, AT1G10170, AT1G12610, AT1G12860, AT1G12980,AT1G13960, AT1G14350, AT1G14920, AT1G15360, AT1G16490, AT1G18570,AT1G19220, AT1G19350, AT1G19850, AT1G21970, AT1G22070, AT1G23420,AT1G24260, AT1G24590, AT1G25560, AT1G26310, AT1G26870, AT1G26945,AT1G27730, AT1G28300, AT1G30210, AT1G30330, AT1G30490, AT1G32330,AT1G32540, AT1G32640, AT1G32770, AT1G33240, AT1G34370, AT1G34790,AT1G35515, AT1G42990, AT1G45249, AT1G46768, AT1G47870, AT1G51700,AT1G52150, AT1G52880, AT1G52890, AT1G53230, AT1G53910, AT1G54060,AT1G55580, AT1G55600, AT1G56010, AT1G56650, AT1G62300, AT1G62360,AT1G63650, AT1G65620, AT1G66350, AT1G66390, AT1G66600, AT1G67260,AT1G68640, AT1G69120, AT1G69180, AT1G69490, AT1G69600, AT1G70510,AT1G71030, AT1G71692, AT1G71930, AT1G73730, AT1G74930, AT1G75080,AT1G76420, AT1G77850, AT1G78600, AT1G79180, AT1G79580, AT1G79840,AT2G01500, AT2G01570, AT2G01930, AT2G02450, AT2G03340, AT2G16910,AT2G17950, AT2G20180, AT2G22300, AT2G22540, AT2G22630, AT2G22770,AT2G23760, AT2G24570, AT2G26150, AT2G27050, AT2G27300, AT2G27990,AT2G28160, AT2G28350, AT2G28550, AT2G28610, AT2G30250, AT2G30432,AT2G33810, AT2G33835, AT2G33860, AT2G33880, AT2G34710, AT2G36010,AT2G36270, AT2G36890, AT2G37260, AT2G37630, AT2G38470, AT2G40220,AT2G40950, AT2G42200, AT2G42830, AT2G43010, AT2G45190, AT2G45660,AT2G46270, AT2G46410, AT2G46680, AT2G46770, AT2G46830, AT2G46870,AT2G46970, AT2G47190, AT2G47460, AT3G01140, AT3G01470, AT3G02990,AT3G03450, AT3G04670, AT3G07650, AT3G10800, AT3G11440, AT3G12250,AT3G13540, AT3G13890, AT3G15170, AT3G15210, AT3G15500, AT3G15510,AT3G16770, AT3G16857, AT3G17609, AT3G18990, AT3G19290, AT3G20310,AT3G20770, AT3G22170, AT3G23130, AT3G23250, AT3G24650, AT3G25710,AT3G26744, AT3G26790, AT3G27785, AT3G27810, AT3G27920, AT3G28470,AT3G28910, AT3G44750, AT3G46640, AT3G48160, AT3G48430, AT3G49940,AT3G50410, AT3G51060, AT3G54220, AT3G54320, AT3G54340, AT3G54620,AT3G55370, AT3G56400, AT3G58070, AT3G58780, AT3G59060, AT3G61850,AT3G61890, AT3G61910, AT3G62420, AT4G00120, AT4G00180, AT4G00220,AT4G01250, AT4G01540, AT4G02560, AT4G04450, AT4G08150, AT4G09820,AT4G09960, AT4G15090, AT4G16110, AT4G16780, AT4G17750, AT4G18960,AT4G20380, AT4G21330, AT4G21750, AT4G23550, AT4G23810, AT4G24020,AT4G24240, AT4G24470, AT4G24540, AT4G25470, AT4G25480, AT4G25490,AT4G25530, AT4G26150, AT4G27330, AT4G27410, AT4G28110, AT4G28610,AT4G30080, AT4G31550, AT4G31800, AT4G31920, AT4G32730, AT4G32880,AT4G32980, AT4G34000, AT4G34590, AT4G34990, AT4G35900, AT4G36730,AT4G36870, AT4G36920, AT4G36930, AT4G37540, AT4G37650, AT4G37750,AT4G38620, AT5G01900, AT5G02030, AT5G02470, AT5G03150, AT5G03680,AT5G03790, AT5G04240, AT5G05410, AT5G06070, AT5G06100, AT5G06650,AT5G06950, AT5G06960, AT5G07100, AT5G07690, AT5G07700, AT5G08130,AT5G09750, AT5G10140, AT5G10510, AT5G11260, AT5G11510, AT5G12870,AT5G13790, AT5G14010, AT5G14750, AT5G14960, AT5G15840, AT5G15850,AT5G16560, AT5G16820, AT5G17300, AT5G17430, AT5G18560, AT5G18830,AT5G20240, AT5G20730, AT5G21120, AT5G22220, AT5G22570, AT5G23000,AT5G23260, AT5G26660, AT5G35550, AT5G35770, AT5G37020, AT5G37260,AT5G40330, AT5G40350, AT5G40360, AT5G41315, AT5G41410, AT5G42630,AT5G43270, AT5G45980, AT5G47220, AT5G48670, AT5G51990, AT5G52830,AT5G53200, AT5G53210, AT5G53950, AT5G54070, AT5G56110, AT5G56270,AT5G56860, AT5G59570, AT5G59820,

AT5G60690, AT5G60890, AT5G60910, AT5G61270, AT5G61420, AT5G61850,AT5G62000, AT5G62020, AT5G62380, AT5G62430, AT5G65050, AT5G66870,AT5G67300, and AT5G67420.

In various embodiments, the engineered protein sensor and/or switch isan engineered version of a Drosophila melanogaster TF, such as CG10325,CG11648, CG6093, CG3796, CG9151, CG15845, CG3935, CG3166, CG8376,CG3258, CG6677, CG3629, CG1034, CG3578, CG11491, CG12653, CG1759,CG6384, CG11924, CG4881, CG8367, CG17894, CG8669, CG2714, CG5893,CG9745, CG5102, CG2189, CG33183, CG9908, CG10798, CG1897, CG11094,CG2711, CG10604, CG32346, CG5714, CG1765, CG7383, CG32180, CG8127,CG1007, CG2988, CG9015, CG14941, CG8365, CG2328, CG8933, CG10488,CG6502, CG10002, CG2707, CG10034, CG2047, CG4059, CG33133, CG9656,CG2692, CG3388, CG7952, CG6494, CG11607, CG9786, CG4694, CG9768, CG1619,CG5748, CG17117, CG17835, CG2275, CG33956, CG10197, CG4717, CG4761,CG3340, CG3647, CG3758, CG4158, CG4148, CG7664, CG10699, CG5954,CG17743, CG1264, CG3839, CG32120, CG1689, CG8346, CG6096, CG8361,CG1705, CG14548, CG8328, CG8333, CG2050, CG18740, CG9045, CG10250,CG11450, CG6534, CG3851, CG1133, CG7467, CG6824, CG5109, CG12212,CG3978, CG17077, CG9610, CG8246, CG6716, CG7230, CG6348, CG10393,CG1849, CG9495, CG1030, CG8544, CG7734, CG1641, CG16738, CG3956, CG3836,CG11121, CG7847, CG3992, CG7938, CG17958, CG6993, CG8573, CG8599,CG8409, CG8068, CG11502, CG4216, CG16778, CG1378, CG6883, CG8651,CG1374, CG1856, CG10619, CG2956, CG10388, CG2762, CG4380, CG6172,CG7803, CG1046, CG1048, CG3411, CG12154, CG7895, CG3827, CG11387,CG17950, CG12287, CG7450, CG2368, CG6143, CG6338, CG2939, CG6464,CG17228, CG1322, CG1449, CG7672, CG14307, CG7771, CG5403, CG3497,CG5488, CG4220, CG2125, CG18412, CG7902, CG7937, CG18023, CG9097,CG2102, CG1130, CG3242, CG10021, CG1132, CG3668, CG11921, CG11922,CG9310, CG8887, CG3114, CG6634, CG1464, CG11049, CG14513, CG3090,CG8404, CG3886, CG12052, CG4354, CG1454, CG7018, CG5583, CG2914, CG4952,CG5683, CG4491, CG33152, CG9930, CG5441, CG6570, CG3905, CG8704,CG17921, CG4817, CG7562, CG2851, CG5965, CG7508, CG5580, CG5557, CG6964,CG5575, CG6794, CG2655, CG3052, CG6545, CG7187, CG17161, CG8625,CG12399, CG1775, CG1429, CG31240, CG7260, CG5529, CG4654, CG12223,CG6376, CG5247, CG11494, CG33261, CG12296, CG8103, CG1072, CG7959,CG7960, CG8567, CG18389, CG11992, CG5069, CG12245, CG10601, CG6103,CG1864, CG2678, CG5264, CG11987, CG6215, CG8522, CG7199, CG11783,CG8396, CG11798, CG9019, CG4029, CG10036, CG7951, CG7659, CG1650,CG10159, CG15319, CG5838, CG9398, CG7413, CG5393, CG10571, CG10605,CG14029, CG6604, CG17888, CG13598, CG4257, CG13951, CG9648, CG11186,CG3858, CG9696, CG5799, CG14938, CG1343, CG6312, CG5201, CG10052,CG8013, CG1447, CG32788, CG11202, CG9415, CG1507, CG10270, CG3998,CG5005, CG10269, CG7391, CG8667, CG8727, CG5206, CG13316, CG7807,CG2819, CG3848, CG16902, CG6269, CG10016, CG7760, CG9653, CG1414,CG15552, CG4013, CG8524, CG1071, CG5649, CG2712, CG1605, CG11182,CG18455, CG4303, CG9102, CG17829, CG2932, CG11551, CG2262, CG8474,CG6352, CG6121, CG7958, CG4143, CG11354, CG5935, CG8290, CG32575,CG9418, CG11352, CG3871, CG6627, CG1024, CG8108, CG2790, CG1966,CG11194, CG9776, CG7758, CG8208, CG2244, CG5067, CG5229, CG18783,CG18124, CG15286, CG11405, CG3268, CG11902, CG5133, CG15269, CG3491,CG17328, CG4185, CG16863, CG12630, CG32904, CG17594, CG1922, CG13906,CG18024, CG9233, CG12690, CG2875, CG17592, CG4136, CG12236, CG3726,CG3815, CG3847, CG14441, CG14438, CG3075, CG4575, CG3032, CG4617,CG9650, CG2116, CG2120, CG2129, CG15336, CG10959, CG18262, CG11294,CG12075, CG15365, CG7041, CG7055, CG2889, CG9817, CG2202, CG11122,CG11696, CG11695, CG11085, CG4404, CG4318, CG15749, CG1716, CG11172,CG11071, CG6211, CG9215, CG8119, CG8944, CG8578, CG8909, CG8924, CG9609,CG6769, CG5927, CG6470, CG7101, CG7556, CG14200, CG9571, CG11710,CG1529, CG11617, CG4133, CG31670, CG11723, CG17257, CG3407, CG17612,CG15435, CG15436, CG9088, CG13775, CG9200, CG4496, CG3838, CG13123,CG18619, CG18144, CG5034, CG12299, CG4621, CG6686, CG6792, CG9932,CG5204, CG9305, CG7099, CG5953, CG17912, CG5545, CG10348, CG10431,CG10446, CG17568, CG10263, CG10366, CG10462, CG10447, CG10631, CG10949,CG9342, CG18362, CG15216, CG1832, CG3136, CG2682, CG1845, CG1621,CG1620, CG1603, CG1602, CG12769, CG11641, CG8643, CG8216, CG1663,CG18446, CG12744, CG1407, CG18011, CG12942, CG12391, CG13204, CG12370,CG8821, CG8819, CG3850, CG4676, CG6061, CG6701, CG17385, CG17390,CG10209, CG8089, CG8092, CG16801, CG8314, CG8388, CG7786, CG4282,CG15710, CG17287, CG18468, CG4903, CG15073, CG11906, CG13424, CG9954,CG10543, CG9437, CG10321, CG10318, CG13493, CG11301, CG10344, CG9895,CG9890, CG9876, CG3941, CG5591, CG3065, CG3328, CG11414, CG4707, CG6905,CG1233, CG17181, CG13897, CG9139, CG2199, CG12104, CG1244, CG15812,CG14962, CG14965, CG12029, CG12605, CG15011, CG5249, CG17334, CG13287,CG13296, CG10274, CG7386, CG10147, CG8591, CG7404, CG7015, CG6683,CG6765, CG5093, CG5187, CG3891, CG3445, CG3654, CG7839, CG6272, CG11799,CG7368, CG4328, CG10704, CG10654, CG14117, CG17361, CG17359, CG7345,CG3919, CG6854, CG13458, CG7372, CG15715, CG9705, CG32171, CG18265,CG7271, CG4076, CG8765, CG11456, CG10565, CG7204, CG11247, CG14451,CG14655, CG14667, CG12162, CG10979, CG10296, CG9727, CG10267, CG33323,CG2702, CG9638, CG7963, CG8145, CG11762, CG8159, CG9793, CG9797, CG8359,CG11966, CG11984, CG11033, CG12952, CG16779, CG8301, CG8319, CG16899,CG8478, CG8484, CG6254, CG4570, CG4820, CG6689, CG6791, CG14710, CG6808,CG14711, CG6813, CG18476, CG6913, CG10042, CG5196, CG5245, CG33976,CG7518, CG15889, CG3143, CG7987, CG14860, CG6654, CG6276, CG5083,CG10278, CG5952, CG10309, CG3995, CG17803, CG17806, CG17802, CG17801,CG7357, CG7785, CG18599, CG7691, CG17186, CG4424, CG4854, CG4413,CG4936, CG4360, CG4217, CG15696, CG5737, CG7056, CG7045, CG7046, CG6990,CG4677, CG33336, CG4374, CG6129, CG5669, CG13617, CG13624, CG6892,CG11375, CG10669, CG4553, CG4730, CG17198, CG17197, CG17195, CG4956,CG32474, CG3350, CG5586, CG1647, CG14514, CG15504, CG15514, CG7928,CG2229, CG12071, CG11317, CG12054, CG1792, CG2052, CG11093, CG11152,CG11153, CG17172, CG6889, CG3743, CG13475, CG3526, CG11398, CG12767,CG15367, CG33473, CG14767, CG3576, CG12659, CG13109, CG12809, CG8817,CG8254, CG16910, CG3274, CG18764, CG32139, CG32577, CG2380, CG15736,CG13399, CG4427, CG12219, CG18647, CG31753, CG33720, CG30011, CG30020,CG30077, CG30401, CG30403, CG30420, CG30431, CG30443, CG31169, CG31224,CG31365, CG31388, CG31392, CG31441, CG31460, CG31481, CG31510, CG31612,CG31632, CG31642, CG31782, CG31835, CG31875, CG31955, CG32006, CG32050,CG32105, CG32121, CG32264, CG32296, CG32532, CG32719, CG32767, CG32772,CG32778, CG32830, CG33695, CG32982, CG33178, CG33213, CG33221, CG33520,CG33525, CG33557, CG33936, CG33980, CG34031, CG12632, CG17469, CG34100,CG34145, CG34149, CG34340, CG34346, CG34367, CG34376, CG34395, CG34403,CG34406, CG34407, CG34415, CG34419, CG34421, CG34422, CG8961, CG9397,CG10037, CG31258, CG31666, CG12196, CG6930, CG12238, CG33546, CG42234,CG34360, CG42267, CG42277, CG42281, CG42311, CG42332, CG42344, CG4807,CG7752, CG12701, CG17100, CG11971, CG42516, CG42515, CG6667, CG1028,CG3281, CG12124, CG42599, CG8506, CG17836, CG1070, and CG8676.

In various embodiments, the engineered protein sensor and/or switch isan engineered version of a mouse TF, such as mouse loci 11538, 11568,11569, 11614, 11622, 11624, 11632, 11634, 11694, 11695, 11733, 11736,11819, 11835, 11859, 11863, 11864, 11865, 11878, 11906, 11908, 11909,11910, 11911, 11920, 11921, 11922, 11923, 11924, 11925, 11991, 12013,12014, 12020, 12021, 12022, 12023, 12029, 12051, 12053, 12142, 12151,12173, 12180, 12189, 12192, 12224, 12265, 12326, 12355, 12387, 12393,12394, 12395, 12399, 12400, 12416, 12417, 12418, 12454, 12455, 12566,12567, 12572, 12578, 12579, 12580, 12581, 12590, 12591, 12592, 12606,12607, 12608, 12609, 12611, 12653, 12677, 12705, 12753, 12785, 12848,12912, 12913, 12914, 12915, 12916, 12951, 13017, 13018, 13047, 13048,13134, 13163, 13170, 13172, 13180, 13196, 13198, 13345, 13390, 13392,13393, 13394, 13395, 13396, 13433, 13435, 13486, 13494, 13496, 13555,13557, 13559, 13560, 13591, 13592, 13593, 13626, 13653, 13654, 13655,13656, 13661, 13709, 13710, 13711, 13712, 13713, 13714, 13716, 13796,13797, 13798, 13799, 13813, 13819, 13864, 13865, 13871, 13872, 13875,13876, 13982, 13983, 13984, 14008, 14009, 14011, 14013, 14025, 14028,14029, 14030, 14055, 14056, 14085, 14105, 14106, 14154, 14155, 14200,14233, 14234, 14235, 14236, 14237, 14238, 14239, 14240, 14241, 14247,14281, 14282, 14283, 14284, 14359, 14390, 14391, 14457, 14460, 14461,14462, 14463, 14464, 14465, 14472, 14489, 14531, 14534, 14536, 14581,14582, 14605, 14632, 14633, 14634, 14659, 14797, 14815, 14836, 14842,14843, 14884, 14885, 14886, 14896, 14912, 15110, 15111, 15161, 15163,15181, 15182, 15183, 15184, 15185, 15193, 15205, 15206, 15207, 15208,15209, 15213, 15214, 15218, 15220, 15221, 15223, 15227, 15228, 15229,15242, 15248, 15251, 15258, 15260, 15273, 15284, 15285, 15331, 15353,15354, 15361, 15364, 15370, 15371, 15372, 15373, 15375, 15376, 15377,15378, 15379, 15384, 15394, 15395, 15396, 15397, 15398, 15399, 15400,15401, 15402, 15403, 15404, 15405, 15407, 15408, 15410, 15412, 15413,15414, 15415, 15416, 15417, 15421, 15422, 15423, 15424, 15425, 15426,15427, 15429, 15430, 15431, 15432, 15433, 15434, 15436, 15437, 15438,15460, 15499, 15500, 15563, 15569, 15900, 15901, 15902, 15903, 15904,15951, 15976, 16150, 16151, 16201, 16348, 16362, 16363, 16364, 16371,16372, 16373, 16391, 16392, 16476, 16477, 16478, 16596, 16597, 16598,16599, 16600, 16601, 16656, 16658, 16761, 16764, 16814, 16815, 16825,16826, 16842, 16869, 16870, 16871, 16872, 16873, 16874, 16875, 16876,16909, 16911, 16917, 16918, 16969, 17095, 17119, 17121, 17122, 17125,17126, 17127, 17128, 17129, 17130, 17131, 17132, 17133, 17134, 17135,17172, 17173, 17187, 17188, 17191, 17192, 17215, 17216, 17217, 17218,17219, 17220, 17257, 17258, 17259, 17260, 17261, 17268, 17274, 17283,17285, 17286, 17300, 17301, 17318, 17341, 17342, 17344, 17354, 17355,17420, 17425, 17428, 17480, 17536, 17537, 17681, 17684, 17692, 17701,17702, 17703, 17749, 17764, 17765, 17859, 17863, 17864, 17865, 17869,17870, 17876, 17877, 17878, 17927, 17928, 17932, 17933, 17936, 17937,17938, 17977, 17978, 17979, 17984, 18002, 18012, 18013, 18014, 18018,18019, 18020, 18021, 18022, 18023, 18024, 18025, 18027, 18028, 18029,18030, 18032, 18033, 18034, 18036, 18037, 18038, 18044, 18045, 18046,18071, 18072, 18088, 18089, 18091, 18092, 18094, 18095, 18096, 18109,18124, 18128, 18129, 18131, 18132, 18140, 18142, 18143, 18171, 18181,18185, 18193, 18198, 18227, 18291, 18292, 18393, 18412, 18420, 18423,18424, 18426, 18432, 18503, 18504, 18505, 18506, 18507, 18508, 18509,18510, 18511, 18514, 18515, 18516, 18519, 18572, 18606, 18609, 18612,18616, 18617, 18626, 18627, 18628, 18667, 18676, 18685, 18736, 18740,18741, 18742, 18771, 18789, 18854, 18933, 18935, 18983, 18985, 18986,18987, 18988, 18990, 18991, 18992, 18993, 18994, 18995, 18996, 18997,18998, 18999, 19009, 19013, 19014, 19015, 19016, 19017, 19018, 19049,19056, 19060, 19084, 19099, 19127, 19130, 19182, 19184, 19202, 19213,19231, 19290, 19291, 19326, 19330, 19377, 19401, 19411, 19434, 19645,19650, 19651, 19664, 19668, 19687, 19696, 19697, 19698, 19708, 19712,19724, 19725, 19726, 19727, 19763, 19820, 19822, 19826, 19883, 19885,20016, 20017, 20018, 20019, 20020, 20021, 20022, 20024, 20128, 20174,20181, 20182, 20183, 20185, 20186, 20204, 20218, 20220, 20230, 20231,20232, 20289, 20371, 20375, 20384, 20409, 20429, 20439, 20464, 20465,20466, 20467, 20471, 20472, 20473, 20474, 20475, 20476, 20480, 20481,20583, 20585, 20586, 20587, 20589, 20591, 20592, 20602, 20613, 20638,20664, 20665, 20666, 20667, 20668, 20669, 20670, 20671, 20672, 20673,20674, 20675, 20677, 20678, 20679, 20680, 20681, 20682, 20683, 20687,20688, 20689, 20728, 20787, 20788, 20807, 20819, 20833, 20841, 20842,20846, 20847, 20848, 20849, 20850, 20851, 20852, 20893, 20901, 20904,20922, 20923, 20924, 20997, 21339, 21340, 21341, 21343, 21349, 21350,21374, 21375, 21380, 21382, 21383, 21384, 21385, 21386, 21387, 21388,21389, 21399, 21400, 21401, 21405, 21406, 21407, 21408, 21410, 21411,21412, 21413, 21414, 21415, 21416, 21417, 21418, 21419, 21420, 21422,21423, 21425, 21426, 21427, 21428, 21429, 21652, 21674, 21676, 21677,21678, 21679, 21685, 21780, 21781, 21783, 21804, 21807, 21815, 21833,21834, 21835, 21843, 21847, 21848, 21849, 21869, 21885, 21886, 21887,21888, 21907, 21908, 21909, 21917, 21929, 21945, 21981, 22025, 22026,22051, 22057, 22059, 22061, 22062, 22088, 22160, 22200, 22221, 22255,22259, 22260, 22278, 22282, 22286, 22326, 22337, 22383, 22385, 22431,22433, 22608, 22632, 22634, 22639, 22640, 22642, 22646, 22654, 22658,22661, 22666, 22668, 22678, 22680, 22685, 22689, 22691, 22694, 22695,22696, 22697, 22698, 22700, 22701, 22702, 22704, 22709, 22710, 22712,22715, 22717, 22718, 22719, 22722, 22750, 22751, 22754, 22755, 22756,22757, 22758, 22759, 22761, 22762, 22764, 22767, 22768, 22770, 22771,22772, 22773, 22775, 22776, 22778, 22779, 22780, 23808, 23827, 23849,23850, 23856, 23857, 23871, 23872, 23885, 23894, 23942, 23957, 23958,23989, 23994, 24068, 24074, 24075, 24113, 24116, 24135, 24136, 26356,26371, 26379, 26380, 26381, 26386, 26404, 26413, 26417, 26419, 26423,26424, 26427, 26461, 26465, 26573, 26754, 26927, 26939, 27049, 27056,27057, 27059, 27081, 27140, 27217, 27223, 27224, 27274, 27386, 28019,29806, 29808, 29813, 29861, 29871, 30046, 30051, 30794, 30841, 30923,30927, 30928, 30942, 30944, 30946, 30951, 50496, 50524, 50721, 50754,50777, 50783, 50794, 50796, 50817, 50868, 50887, 50907, 50913, 50914,50916, 50996, 51792, 51813, 52024, 52040, 52231, 52502, 52609, 52615,52705, 52708, 52712, 52897, 53314, 53317, 53357, 53380, 53415, 53417,53626, 53868, 53869, 53970, 53975, 54006, 54123, 54131, 54132, 54139,54169, 54343, 54352, 54388, 54422, 54446, 54562, 54601, 54633, 54678,54711, 55927, 55942, 55994, 56030, 56070, 56196, 56198, 56218, 56220,56222, 56233, 56275, 56309, 56312, 56314, 56321, 56353, 56380, 56381,56404, 56406, 56449, 56458, 56469, 56484, 56490, 56501, 56503, 56505,56522, 56523, 56525, 56613, 56642, 56707, 56736, 56771, 56784, 56787,56805, 56809, 56856, 56869, 57080, 57230, 57246, 57314, 57316, 57376,57737, 57745, 57748, 57756, 57765, 57782, 58172, 58180, 58198, 58202,58206, 58234, 58805, 59004, 59021, 59024, 59026, 59035, 59057, 59058,60345, 60406, 60611, 64050, 64144, 64290, 64379, 64383, 64384, 64406,64453, 64685, 65020, 65247, 65255, 65256, 65257, 66056, 66118, 66136,66213, 66233, 66277, 66352, 66376, 66420, 66464, 66491, 66505, 66556,66596, 66622, 66634, 66642, 66671, 66698, 66729, 66799, 66867, 66880,66923, 66930, 66959, 66970, 66980, 66985, 67057, 67065, 67122, 67150,67151, 67155, 67199, 67235, 67260, 67279, 67288, 67367, 67370, 67379,67381, 67389, 67419, 67439, 67575, 67657, 67673, 67692, 67710, 67815,67847, 67873, 67949, 67985, 67993, 68040, 68153, 68196, 68268, 68346,68479, 68558, 68701, 68705, 68776, 68839, 68842, 68854, 68910, 68911,68992, 69020, 69125, 69167, 69168, 69188, 69234, 69241, 69257, 69260,69299, 69317, 69389, 69539, 69606, 69656, 69716, 69790, 69833, 69890,69920, 69944, 70073, 70122, 70127, 70315, 70350, 70392, 70408, 70428,70459, 70497, 70508, 70601, 70625, 70637, 70650, 70673, 70779, 70796,70797, 70823, 70859, 70981, 71041, 71063, 71131, 71137, 71163, 71176,71241, 71280, 71371, 71375, 71409, 71458, 71468, 71592, 71597, 71702,71722, 71752, 71767, 71777, 71782, 71793, 71828, 71834, 71838, 71839,71939, 71949, 71990, 71991, 72057, 72074, 72135, 72180, 72195, 72199,72290, 72293, 72323, 72325, 72388, 72459, 72465, 72475, 72556, 72567,72615, 72720, 72727, 72739, 72823, 72949, 72958, 73178, 73181, 73340,73389, 73451, 73469, 73503, 73610, 73614, 73844, 73845, 73945, 74007,74068, 74106, 74120, 74123, 74149, 74164, 74168, 74197, 74282, 74318,74322, 74326, 74335, 74352, 74377, 74481, 74533, 74561, 74570, 74838,75196, 75199, 75210, 75291, 75305, 75339, 75387, 75480, 75482, 75507,75572, 75599, 75605, 75646, 75725, 75901, 76007, 76022, 76294, 76308,76365, 76389, 76467, 76572, 76580, 76793, 76803, 76804, 76834, 76893,76900, 77057, 77114, 77117, 77264, 77286, 77318, 77480, 77683, 77889,77907, 77913, 78020, 78088, 78246, 78251, 78284, 78455, 78469, 78541,78619, 78656, 78699, 78703, 78783, 78829, 78910, 78912, 78921, 78929,79221, 79233, 79362, 79401, 80283, 80509, 80720, 80732, 80859, 80902,81601, 81630, 81703, 81845, 81879, 83383, 83395, 83396, 83557, 83602,83925, 83993, 84653, 93674, 93681, 93686, 93691, 93759, 93760, 93761,93762, 93837, 93871, 94047, 94112, 94187, 94275, 96979, 97064, 97165,98053, 98403, 99377, 99730, 100090, 100563, 100710, 100978, 101095,101206, 102162, 102209, 102334, 103136, 103806, 103889, 104328, 104349,104360, 104383, 104384, 104394, 104886, 105377, 105594, 105859, 106795,106894, 107351, 107433, 107499, 107503, 107568, 107586, 107751, 107765,107771, 107889, 107932, 107951, 108060, 108098, 108143, 108655, 108672,108857, 109113, 109115, 109575, 109594, 109663, 109676, 109889, 109910,109958, 109972, 109973, 109995, 110052, 110061, 110068, 110109, 110147,110506, 110521, 110616, 110641, 110647, 110648, 110784, 110796, 110805,110913, 112077, 114142, 114565, 114606, 114642, 114774, 114889, 116810,116848, 116870, 116871, 116912, 117168, 117198, 117590, 118445, 140477,140490, 140500, 140577, 140743, 170574, 170644, 170729, 170740, 170767,170787, 170791, 170826, 170938, 192195, 192231, 192285, 192651, 192657,193796, 195333, 208076, 208258, 208266, 208292, 208439, 208677, 208715,209011, 209357, 209361, 209416, 209446, 209448, 209707, 210135, 210162,211378, 212168, 212276, 212391, 212712, 213010, 213990, 214105, 214162,214384, 214669, 214899, 215031, 216151, 216154, 216285, 216456, 216558,216578, 217031, 217082, 217127, 217166, 217558, 218030, 218440, 218490,218624, 218772, 218989, 219150, 223227, 223690, 223701, 223922, 224419,224585, 224656, 224694, 224829, 224902, 224903, 225876, 225895, 225998,226049, 226182, 226442, 226641, 226747, 226896, 227099, 227644, 227656,227940, 228136, 228598, 228731, 228775, 228790, 228829, 228839, 228852,228869, 228876, 228880, 228980, 229004, 229534, 229663, 229906, 230073,230162, 230587, 230674, 230700, 230753, 230908, 230936, 230991, 231044,231051, 231329, 231386, 231986, 231991, 232232, 232337, 232807, 232853,232854, 232878, 232906, 233056, 233410, 233490, 233863, 233887, 233890,233908, 233987, 234725, 234959, 235028, 235041, 235050, 235320, 235442,235582, 235623, 235682, 236193, 237052, 237336, 237409, 237615, 237758,237960, 238247, 239099, 239546, 239652, 240064, 240120, 240263, 240427,240442, 240476, 240590, 240690, 241066, 241447, 241520, 242523, 242620,242705, 243187, 243833, 243906, 243931, 243963, 243983, 244349, 244713,244813, 244954, 245572, 245583, 245596, 245688, 245841, 246086, 246196,246198, 246791, 252829, 260298, 268281, 268301, 268396, 268448, 268564,268741, 268903, 268932, 269252, 269713, 269870, 270076, 270627, 271278,271305, 272347, 272359, 272382, 277353, 319196, 319207, 319535, 319594,319599, 319601, 319615, 319695, 319785, 320067, 320376, 320429, 320586,320595, 320790, 320799, 320875, 320995, 328572, 330301, 330361, 330502,332937, 338353, 347691, 353187, 353208, 378435, 381319, 386626, and386655.

Illustrative aTFs are found in Ramos, et al. Microbiology and MolecularBiology Reviews, June 2005, p. 326-356 and Tropell, et al. Microbiol MolBiol Rev. 2004 September; 68(3):474-500, the contents of which arehereby incorporated by reference in their entireties.

Protein sensor and/or switch amino acid sequences upon which engineeringis to occur may, in various embodiments, be selected by sequencehomology using one or more of BLASTP, PSI-BLAST, DELTA-BLAST, OR HMMER,JackHMMER, or the corresponding nucleotide sequences selected bysequence homology search.

Methods of identifying protein sequences that can be candidate proteinsensors and/or switches are found in US 2016/0063177, the entirecontents of which are hereby incorporated by reference in its entirety.

Various protein sensor and/or switches are engineered as part of theinvention and can be interrogated with target molecules (cellularly oracellularly). Illustrative engineering approaches include mutagenesisthat alters the binding activity of an allosteric protein, e.g. makingthe allosteric protein suitable for binding the target molecule at theexpense of the allosteric proteins cognate ligand (i.e. the ligand thatbinds to the wild type allosteric protein). In some embodiments,mutagenesis comprises introducing one or more amino acid mutations, e.g.independently selected from substitutions, insertions, deletions, andtruncations.

In some embodiments, the amino acid mutations are amino acidsubstitutions, and may include conservative and/or non-conservativesubstitutions.

“Conservative substitutions” may be made, for instance, on the basis ofsimilarity in polarity, charge, size, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the amino acid residuesinvolved. The 20 naturally occurring amino acids can be grouped into thefollowing six standard amino acid groups: (1) hydrophobic: Met, Ala,Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3)acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influencechain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

As used herein, “conservative substitutions” are defined as exchanges ofan amino acid by another amino acid listed within the same group of thesix standard amino acid groups shown above. For example, the exchange ofAsp by Glu retains one negative charge in the so modified polypeptide.In addition, glycine and proline may be substituted for one anotherbased on their ability to disrupt α-helices.

As used herein, “non-conservative substitutions” are defined asexchanges of an amino acid by another amino acid listed in a differentgroup of the six standard amino acid groups (1) to (6) shown above.

In various embodiments, the substitutions may also include non-classicalamino acids (e.g. selenocysteine, pyrrolysine, N-formylmethionineβ-alanine, GABA and δ-Aminolevulinic acid, 4-aminobenzoic acid (PABA),D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu,ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline,sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, 6-alanine,fluoro-amino acids, designer amino acids such as β methyl amino acids, Cα-methyl amino acids, N α-methyl amino acids, and amino acid analogs ingeneral).

The present invention pertains to various target molecules, for which aprotein sensor and/or switch may be engineered. Illustrative targetmolecules include one or more of the compounds described in WO2015/017866, e.g. at paragraphs [00107]-[00112], the entire contents ofwhich are hereby incorporated by reference in its entirety. In variousembodiments, the various target molecules of the invention are toxic toa cell and/or cannot be readily bind or interact with a protein sensorand/or switch in a detectable manner in a cellular environment. Invarious embodiments, the protein sensor and/or switch is selected basedon its cognate ligand identity and any commonality the cognate ligandmay have with a target molecules. For example, a shared chemical groupbetween a cognate ligand and a target molecule may direct one to theprotein sensor and/or switch which binds to the cognate ligand and leadto the engineering of the protein sensor and/or switch so it can bind tothe target molecule.

In some embodiments, the present invention relates to antibiotics. Tocircumvent toxicity of antibiotics, various resistance mechanisms may beintroduced into a producing cell. Without limitation, these may includeenzymes which degrade or chemically render the antibiotic less toxic tothe producing cell. Resistance to the antibiotics mechanism of actionmay be conferred by alterations introduced into the cellular context ofthe producing cell. For instance, the ribosome may be altered to avoidantibiotic binding and relieve inhibition of protein synthesis. A cellwall biosynthetic enzyme may be mutated to ablate antibiotic binding andrelieve inhibition of cell wall biosynthesis. A pump which lowers theintracellular concentration may be expressed. A specific antibioticbinding protein may be expressed.

In some embodiments, the target molecule is an antibiotic (e.g. onewhich is lethal to a host cell). In some embodiments, the antibiotic isa beta-lactam antibiotic, such as a penicillin, e.g., Penicillin,Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin,Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin,Oxacillin, Penicillin G, Penicillin V, Piperacillin, Penicillin G,Temocillin, Ticarcillin. In some embodiments, the antibiotic is anAminoglycoside, e.g., Amikacin, Gentamicin, Kanamycin, Neomycin,Netilmicin, Tobramycin, Paromomycin, Streptomycin, or Spectinomycin. Insome embodiments, the antibiotic is an Ansamycin, e.g., Geldanamycin,Herbimycin, or Rifaximin. In other embodiments, the antibiotic is apenem such as faropenem or Ritipenem; or a Carbacephem such asLoracarbef; or a carbapenem such as Ertapenem, Doripenem,Imipenem/Cilastatin, or Meropenem. In other embodiments, the antibioticis an Cephalosporin, e.g., Cefadroxil, Cefazolin, Cefalotin orCefalothin, Cefalexin (or cephalexin), Cefaclor, Cefamandole, Cefoxitin,Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone,Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime,Ceftriaxone (IV and IM), Cefepime, Ceftaroline fosamil, Ceftobiprole,Ceftiofur, Cefquinome, or Cefovecin. In yet other embodiments, theantibiotic is a p-lactamase inhibitor, such as, for example, Penam(Sulbactam Tazobactam), Clavam (Clavulanic acid), Avibactam, orVaborbactam. In other embodiments, the antibiotic is a glycopeptide suchas Teicoplanin, Vancomycin, Telavancin, Dalbavancin, or Oritavancin. Insome embodiments, the antibiotic is a lincosamides such as, e.g.,Clindamycin or Lincomycin. In yet other embodiments, the antibiotic is alipopeptide such as Daptomycin. In some embodiments, the antibiotic is aMacrolide such as, e.g., Azithromycin, Clarithromycin, Dirithromycin,Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, orSpiramycin. In some embodiments, the antibiotic is a Monobactam such asAztreonam, Tigemonam, Carumonam, or Nocardicin A. In some embodiments,the antibiotic is a nitrofuran, such as, e.g., Furazolidone orNitrofurantoin. In some other embodiments, the antibiotic is anoxazolidinones such as, e.g., Linezolid, Posizolid, Radezolid, orTorezolid. In other embodiments, the antibiotic is a polypeptide, suchas Bacitracin, Colistin, or Polymyxin B. In yet other embodiments, theantibiotic is a Quinolone or Fluoroquinolone such as, e.g.,Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin,Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin,Trovafloxacin, Grepafloxacin, Sparfloxacin, or Temafloxacin. In someembodiments, the antibiotic is a sulfonamide such as Mafenide,Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine,Sulfamethizole, Sulfamethoxazole, Sulfanilimide (archaic),Sulfasalazine, Sulfisoxazole, Trimethoprim,Trimethoprim-Sulfamethoxazole(Co-trimoxazole) (TMP-SMX), orSulfonamidochrysoidine. In some embodiments, the antibiotic is aTetracycline, e.g., Demeclocycline, Doxycycline, Minocycline,Oxytetracycline, or Tetracycline. In some embodiments, the antibiotic isa drug against mycobacteria, such as Clofazimine, Dapsone, Capreomycin,Cycloserine, Ethambutol(Bs), Ethionamide, Isoniazid, Pyrazinamide,Rifampicin (Rifampin in US), Rifabutin, Rifapentine, Streptomycin. Insome embodiments, the antibiotic is Arsphenamine, Chloramphenicol(Bs),Fosfomycin, Fusidic acid, Metronidazole, Mupirocin, Platensimycin,Quinupristin/Dalfopristin, Thiamphenicol, Tigecycline(Bs), Tinidazole.In yet other embodiments the antibiotic is teixobactin, or relatedmolecules in this new class of antibiotics, which harm bacteria bybinding lipid II and/or lipid III, which are important cell wallprecursors.

Illustrative protein sensors and/or switches and cognate ligands arefound in WO 2015/127242, for instance in the table of page 7, thecontents of which are hereby incorporated by reference in theirentirety.

In various embodiments, the protein sensor and/or switch is anengineered using design from existing allosteric proteins, e.g. aTFs. Invarious embodiments, the designing comprises in silico design.Illustrative design principles are found in US 2016/0063177, the entirecontents of which are hereby incorporated by reference in theirentirety.

For example, in various embodiments, molecular modeling is used topredict mutations in an allosteric protein which may render theallosteric protein able to bind one or more target molecules. In variousembodiments, reference to an experimentally derived three-dimensionalprotein structure, typically obtained through experimental methodsincluding, but not limited to, x-ray crystallography, nuclear magneticresonance (NMR), scattering, or diffraction techniques, is employed tomodel and/or predict mutations in an allosteric protein which may renderthe allosteric protein able to bind one or more target molecule. Invarious embodiments, the ROSETTA software suite is employed to assistwith modelling (see Kaufmann et al. Biochemistry. 2010 Apr. 13;49(14):2987-98, the entire contents of which are hereby incorporated byreference in its entirety). Alternatively, or in combination, a homologymodeling algorithm such as ROBETTA, TASSER, I-TASSER, HHpred, HHsearch,or MODELLER, or SWISS-MODEL can be used. In some embodiments, such as(without limitation) those in which allosteric protein lacks anexperimentally derived three-dimensional protein structure, a homologymodeling algorithm can be used to build the sequence homology models. Invarious embodiments, one or more sequence or structural homologs haveless than 90% amino acid sequence identity, less than 85% amino acidsequence identity, less than 80% amino acid sequence identity, less than75% amino acid sequence identity, less than 70% amino acid sequenceidentity, less than 65% amino acid sequence identity, less than 60%amino acid sequence identity, less than 55% amino acid sequenceidentity, less than 50% amino acid sequence identity, less than 45%amino acid sequence identity, less than 40% amino acid sequenceidentity, less than 35% amino acid sequence identity, less than 30%amino acid sequence identity, less than 25% amino acid sequenceidentity, or less amino acid sequence identity to the amino acidsequence of the three-dimensional protein structure. Illustrativehomology modelling methods and principles are found in US 2016/0063177,e.g. at paragraphs [0085]-[0093], the entire contents of which arehereby incorporated by reference in its entirety.

In some embodiments, a structure of an allosteric protein is evaluatedfor alterations which may render the allosteric protein able to bind oneor more target molecules (e.g. by docking a one or more target moleculesinto the structure of an allosteric protein). Illustrative dockingmethods and principles are found in US 2016/0063177, e.g. at paragraphs[0095]-[0101], the entire contents of which are hereby incorporated byreference in its entirety.

In various embodiments, libraries of potential mutations to theallosteric protein are made and selection, positive or negative, is usedto screen desired mutants.

In various embodiments, engineering may use the technique ofcomputational protein design (as disclosed in U.S. Pat. No. 7,574,306and U.S. Pat. No. 8,340,951, which are hereby incorporated by referencein their entirety) directed evolution techniques, rational mutagenesis,or any suitable combination thereof.

In other embodiments, mutation techniques such as gene shuffling,homologous recombination, domain swapping, deep mutation scanning,and/or random mutagenesis may be employed.

In various embodiments, the following table provides illustrativesensors that may be designed in accordance with various embodiments ofthe present invention. For instance, in various embodiments, one mayselect an aTF (“Chassis”) and/or native ligand and make reference to aprovided representative structure (PDB) to, in accordance with thedisclosure herein, design a senor to a target molecule or class oftarget molecules (see Target Molecule Property column).

TABLE 1 aTF Representative Target Molecule (“Chassis”) Native LigandNative Host Structure (PDB) Property QscR Bound to N-3-oxo- Psudemonas3SZT long chain fatty acids and dodecanoyl-L- aeruginosa homoserinelactones Homoserine Lactone NtcA 2-oxoglutarate, Anabaena 3LA2, LA3,3LA7 3-7 carbon acids/ 2,2- cyanobacterium alcohols difluoropentanoicacid CarH adenosylcobalamin Thermus 5C8A, 5C8D, 5C8E, cobalaminethermophilus 5C8F CcpN ADP Bacillus subtilis 3FV6, 3FWR, 3FWSnucleotides, nucleosides repressor BtAraR arabinose Bacteriodes 5BS6,5DD4, 5DDG, saccharides thetaiotaomicron 5DEQ AraR arabinose Bacteroides5BS6, 5DD4, 5DDG, saccharides thetaiotaomicron VPI 5DEQ AhrR ArginineBacillus subtilis 2P5L 2P5M charged amino acids, quanidino groupsRv1846c betalactams Mycobacterium 2G9W betalactams tuberculosis. CviR C6HSL Chromobacterium 3QP1, 3QP2, 3QP4, short chain fatty acids violaceum3QP5, 3QP6, 3QP8 and homoserine lactones MtbCRP cAMP Myco tuberculosis3I54 cyclic nucleotides BmrR cationic antibiotics, Bacillus subtilis3Q1M, 3Q2Y, 3Q3D, cationic multirings dyes, and 3Q5P, 3Q5R, 3Q5Sdisinfectants Rrf2 cysteine Bacillus subtilis 2Y75 hydrophobic aminoacids, sulfur containing molecules CGL2612 drugs Corynebacterium 1V7B,2ZOY rigid multiring molecules glutamicum TtgR drugs Pseudomonas 2UXH,2UXI, 2UXO, rigid multiring molecules putida 2UXP, 2UXU QacR Ethidium,Staphylococcus 3BR3 3BR6 2DTZ chemically rigid, bivalent rhodamine,Aureus 2HQ5 compounds. Cra fructose 1 Pseudomonas 3O74, 3O75 sugarphosphates phosphate putida GabR gamma- Bacillus subtilis 4N0B shortchain amines and aminobutyric acid acids YvoA glucosamine-6- Bacillussubtilis 4U0V, 4U0W, 4U0Y, C5, C6 sugars phosphate, 4WWCacetylglucosamine- 6-phosphate CggR glucose-6- Bacillus subtilis 2OKG,3BXE, 3BXF, C5, C6 sugars phosphate and 3BXG, 3BXH fructose-6- AlsoCited By: 4OQP, phosphate 4OQQ CodY GTP, Isoleucine Bacillus subtilis2B0L, 2B18, 2GX5, hydrophobic amino acids 2HGV nucleosids, nucleotides,nucleotide phosphates HrcA heat Thermotoga 1STZ temperature, useful formaritima circular permutation/stability measurements RovA heat Yersiniapestis 4AIH, 4AIJ, 4AIK temperature, useful for circularpermutation/stability measurements LldR lactose Corynebacterium 2DI3saccharides (CGL2915) glutamicum LacI Lactose/IPTG E. coli 2p9hsaccharides NMB0573/ leucine methionine Neisseria 2P5V, 2P6S, 2P6Thydrophobic amino acids, AsnC meningitidis sulfer containing compoundsFapR malonyl-CoA Bacillus subtilis 2F3X, 2F41 c3-c7 molecules, CoAcofactors FapR malonyl-CoA Staphylococcus 4A0X, 4A0Y, 4A0Z, c3-c7molecules, CoA Aureus 4A12 cofactors LmrR MDR pump Lactococcus lactis3F8B, 3F8C, 3F8F rigid multiring molecules controller SMET MDR pumpStenotrophomonas 2W53 rigid multiring molecules controller maltophiliaSCO4008 methylene blue, Streptomyces 2D6Y crystal coelicolorvioletcationic antibiotics, dyes, and disinfectants MntR Mn2+ Bacillussubtilis 4hv6 metals and cations Rex NADH Bacillus subtilis, 2VT2, 2VT3cofactors Thermus thermophilus, Thermus aquaticus NikR Nickle Helobacterpylori 3PHT, 3QSI, 2WVB DNR NO (via heme) Pseudomonas 2Z69 metals andcations aeruginosa FadR oleoyl-CoA Vibrio cholerae 4P96, 4P9U, 4PDK longchain fatty acids and cofactors MosR oxidative state Mycobacterium 4FX0,4FX4 oxidative state, useful for tuberculosis. circular permutation OhrRoxidative state Bacillus subtilis 1Z91, 1Z9C oxidative state, useful for(cys) circular permutation SarZ oxidative stress Staphylococcus 3HRM,3HSE, 3HSR oxidative state, useful for Aureus circular permutation TsaRpara- Comamonas 3FXQ, 3FXR, 3FXU, c6-c12 aromatics toluensulfonatetestosteroni 3FZJ HetR PatS Anabaena sp. 4YNL, 4YRV peptides andproteins NprR peptide Bacillus thuringiensis 4GPK peptides and proteinsMexR peptide Pseudomonas 3ECH peptides and proteins aeruginosa PhoP PhoRMycobacterium 2PMU peptides and proteins tuberculosis. PurRPhosphoribosyl- Bacillus subtilis 1P4A phosphorilated sugarspyrophosphate PcaV protocatechuate (a Streptomyces 4FHT, 4G9Y aromaticacids, c4-c10 (SCO6704) dihyroxy benzoic coelicolor acids acid) DesRself His-PO4 Bacillus subtilis 4LDZ, 4LE0, 4LE1, useful for circular4LE2 permutation SinR sinL dimer? Bacillus subtilis 2YAL, 3QQ6 peptidesand proteins EthR something Mycobacterium 1T56 c4-c20 hydrophobichydrophobic tuberculosis. molecules BlcR succinate Agrobacterium 3MQ0short chain aldehydes semialdehyde tumefaciens TetR-class TetPasteurella 2VPR rigid multiring molecules H multocida TetR TetracyclineE. coli Tn10 4AC0 rigid multiring molecules TreR trehalose Bacillussubtilis 2OGG saccharides DntR TsaR type LTTR Burkholderia cepacia 5AE3,5AE4 c6-c12 aromatics HyIIIR unknown large Bacillus cereus 2FX0 largemoledules molecule CprB γ-butyrolactones Streptomyces 4PXI short chainlactones coelicolor AcuR acrylic acid Rhodobacter 3BRU Short chain acidand sphaeroides hydrocarbons

In various embodiments, the amino acids targeted for mutation or insilico design are those within about 3, or about 5, or about 7, or about10, or about 12 Angstroms (e.g. between about 3 to about 12 Angstroms,or between about 5 to about 12 Angstroms, or between about 7 to about 12Angstroms, or between about 10 to about 12 Angstroms, or between about 3to about 5 Angstroms, or between about 3 to about 7 Angstroms, orbetween about 3 to about 10 Angstroms) of a ligand modeled into abinding pocket either through docking or by experimental methods such asX-ray crystallography.

Mutated allosteric proteins that may be protein sensors and/or switchesable to bind one or more target molecules can be screen using standardbinding assays (e.g. fluorescent, radioactive assays, etc.).

In various embodiments, the protein sensor and/or switch is engineeredas described in Taylor, et al. Nat. Methods 13(2): 177, the entirecontents of which are hereby incorporated by reference in its entirety.

In various embodiments, the host cells of the present invention includeeukaryotic and/or prokaryotic cells, including bacterial, yeast, algal,plant, insect, mammalian cells (human or non-human), and immortal celllines.

For example, the host cell may be Escherichia coli, Saccharomycescerevisiae, Pichia pastoris, Saccharomyces castellii, Kluyveromyceslactis, Pichia stipitis, Schizosaccharomyces pombe, Chlamydomonasreinhardtii, Arabidopsis thaliana, or Caenorhabditis elegans. In someembodiments the host cell is a bacterial cell, such as Escherichia spp.,Streptomyces spp., Zymonas spp., Acetobacter spp., Citrobacter spp.,Synechocystis spp., Rhizobium spp., Clostridium spp., Corynebacteriumspp., Streptococcus spp., Xanthomonas spp., Lactobacillus spp.,Lactococcus spp., Bacillus spp., Pedobacter spp., Bacteroides spp.,Alcaligenes spp., Pseudomonas spp., Aeromonas spp., Azotobacter spp.,Comamonas spp., Mycobacterium spp., Rhodococcus spp., Gluconobacterspp., Ralstonia spp., Acidithiobacillus spp., Microlunatus spp.,Geobacter spp., Geobacillus spp., Arthrobacter spp., Flavobacteriumspp., Serratia spp., Saccharopolyspora spp., Thermus spp.,Stenotrophomonas spp., Chromobacterium spp., Sinorhizobium spp.,Saccharopolyspora spp., Agrobacterium spp. and Pantoea spp. Thebacterial cell can be a Gram-negative cell such as an E. coli, or aGram-positive cell such as a species of Bacillus.

In other embodiments, the cell is a fungal cell such as a yeast cell,e.g., Saccharomyces spp., Schizosaccharomyces spp., Pichia spp., Paffiaspp., Kluyveromyces spp., Candida spp., Talaromyces spp., Brettanomycesspp., Pachysolen spp., Debaryomyces spp., Yarrowia spp., and industrialpolyploid yeast strains. Preferably the yeast strain is a S. cerevisiaestrain or a Yarrowia spp. strain. Other examples of fungi includeAspergillus spp., Pennicilium spp., Fusarium spp., Rhizopus spp.,Acremonium spp., Neurospora spp., Sordaria spp., Magnaporthe spp.,Allomyces spp., Ustilago spp., Botrytis spp., and Trichoderma spp.

In other embodiments, the cell is an algal cell or a plant cell (e.g.,A. thaliana, C. reinhardtii, Arthrospira, P. tricomutum, T. suecica, P.carterae, P. tricomutum, Chlorella spp., such as Chlorella vulgaris).

Target cells can include transgenic and recombinant cell lines. Inaddition, heterologous cell lines can be used, such as Chinese HamsterOvary cells (CHO).

In some embodiments, the host cell is an Actinomycetes spp. cell.Actinomycetes are a heterogeneous collection of bacteria that formbranching filaments which include, for example, Actinomyces,Actinomadura, Nocardia, Streptomyces and related genera. In someembodiments, Actinomyces comprise Streptomyces. In some embodiments, theActinomycetes spp. cell is a Streptomyces cell. (e.g. S. coelicolor).Streptomyces include, by way of non-limiting example, S. noursei, S.nodosus, S. natalensis, S. venezuelae, S. roseosporus, S. fradiae, S.lincolnensis, S. alboniger, S. griseus, S. rimosus, S. aureofaciens, S.clavuligerus, S. avermitilis, S. platensis, S. verticillus, S.hygroscopicus, and S. viridochromeogenes.

In some embodiments, the host cell is a Bacillus spp. cell. In someembodiments, the Bacillus spp. cell is selected from B. alcalophilus, B.alvei, B. aminovorans, B. amyloliquefaciens, B. aneurinolyticus, B.anthracis, B. aquaemaris, B. atrophaeus, B. boroniphilus, B. brevis, B.caldolyticus, B. centrosporus, B. cereus, B. circulans, B. coagulans, B.firmus, B. flavothermus, B. fusiformis, B. galliciensis, B. globigii, B.infernus, B. larvae, B. laterosporus, B. lentus, B. licheniformis, B.megaterium, B. mesentericus, B. mucilaginosus, B. mycoides, B. natto, B.pantothenticus, B. polymyxa, B. pseudoanthracis, B. pumilus, B.schlegelii, B. sphaericus, B. sporothermodurans, B. stearothermophilus,B. subtilis, B. thermoglucosidasius, B. thuringiensis, B. vulgatis, andB. weihenstephanensis.

In various embodiments, the nucleic acid is provided to host cell by oneor more of by electroporation, chemical transformation, ballistictransformation, pressure induced transformation, electrospray injection,mechanical shear forces induced, for example, in microfluids, and carbonnanotubes, nanotube puncture, induced natural competence mechanisms ofan organism, merging of protoplasts, and conjugation with Agrobacterium.

In vitro transcription, i.e. the in vitro synthesis of single-strandedRNA molecules, is a routine laboratory procedure. While variations inthe methodology are possible, the same basic procedure is followed inmost in vitro transcription protocols. Specifically, one prepares a DNAtemplate corresponding to the sequence of interest. To allow run offtranscription, plasmid DNA template is generally linearized with arestriction enzyme. In addition to plasmid DNA, PCR products andsynthetic oligonucleotides, among others, can be used as templates fortranscription reactions. The template DNA is then transcribed by an RNApolymerase, e.g. T7, T3 or SP6 RNA phage polymerase, in the presence ofribonucleoside triphosphates (rNTPs). The polymerase traverses thetemplate strand and uses base pairing with the DNA to synthesize acomplementary RNA strand (using uracil in the place of thymine). The RNApolymerase travels from the 3→5′ end of the DNA template strand, toproduce an RNA molecule in the 5→3′ direction. Further details areavailable in Rio, et al. RNA: A Laboratory Manual. Cold Spring Harbor:Cold Spring Harbor Laboratory Press, 2011, 205-220, the contents ofwhich are hereby incorporated by reference in their entirety.

The most frequently used in vitro or cell-free translation systemsconsist of extracts from a biological source, e.g. rabbit reticulocytes,wheat germ, HeLa, and E. coli. All are typically prepared as crudeextracts containing all the macromolecular components (e.g. 70 S or 80 Sribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation, elongation andtermination factors, etc.) required for translation of exogenous RNA.Extracts may be supplemented with amino acids, energy sources (e.g. ATP,GTP), energy regenerating systems (e.g. creatine phosphate and creatinephosphokinase for eukaryotic systems, and phosphoenol pyruvate andpyruvate kinase for the E. coli lysate), and other co-factors (e.g. Me,etc.).

In various embodiments, the present invention employs “coupled” or“linked” IVTT. In various embodiments, the present invention employsIVTT in which the transcription and translation are not coupled, i.e.separate.

There are two approaches to in vitro protein synthesis based on thestarting genetic material i.e. RNA or DNA. Standard translation systems,such as reticulocyte lysates and wheat germ extracts, use RNA as atemplate; whereas “coupled” and “linked” systems start with DNAtemplates, which are transcribed into RNA then translated. Either issuitable for use in the invention described herein.

Rabbit reticulocyte lysate is a highly efficient in vitro eukaryoticprotein synthesis system used for translation of exogenous RNAs (eithernatural or generated in vitro). In vivo, reticulocytes are highlyspecialized cells primarily responsible for the synthesis of hemoglobinand these immature red cells have adequate mRNA, as well as completetranslation machinery, for extensive globin synthesis. The endogenousglobin mRNA is eliminated by incubation with a nuclease, e.g. aCat²⁺dependent micrococcal nuclease, which is later inactivated, e.g. bychelation of the Cat²⁺ by, for example, EGTA. Untreated reticulocytelysate translates endogenous globin mRNA, exogenous RNAs, or both. Thistype of lysate is typically used for studying the translation machinery,e.g. studying the effects of inhibitors on globin translation. Both theuntreated and treated rabbit reticulocyte lysates have low nucleaseactivity and are capable of synthesizing a large amount of full-lengthproduct. Both lysates are appropriate for the synthesis of largerproteins from either capped or uncapped RNAs.

Wheat germ extract has low background incorporation due to its low levelof endogenous mRNA. Wheat germ lysate efficiently translates exogenousRNA from a variety of different organisms. Both reticulocyte and wheatgerm extracts translate RNA isolated from cells and tissue or thosegenerated by in vitro transcription. When using RNA synthesized invitro, the presence of a 5′ cap structure may enhance translationalactivity. Typically, translation by wheat germ extract is morecap-dependent than translation by reticulocyte extracts. If capping ofthe RNA is impossible and the protein yield from an uncapped mRNA islow, the coding sequence can be subcloned into a prokaryotic vector andexpressed directly from a DNA template in an E. coli cell-free system.

E. coli cell-free systems consist of a crude extract that is rich inendogenous mRNA. The extract is incubated during preparation so thatthis endogenous mRNA is translated and subsequently degraded. Becausethe levels of endogenous mRNA in the prepared lysate is low, theexogenous product is easily identified. In comparison to eukaryoticsystems, the E. coli extract has a relatively simple translationalapparatus with less complicated control at the initiation level,allowing this system to be very efficient in protein synthesis. E. coliare particularly suited for coupled transcription:translation from DNAtemplates.

In standard translation reactions, purified RNA is used as a templatefor translation. Linked or coupled systems, on the other hand, use DNAas a template. RNA is transcribed from the DNA and subsequentlytranslated without any purification. Such systems typically combine aprokaryotic phage RNA polymerase and promoter (T7, T3, or SP6) witheukaryotic or prokaryotic extracts to synthesize proteins from exogenousDNA templates. DNA templates for IVT or IVTT reactions may be clonedinto plasmid vectors or generated by PCR. The linked or coupled systemis a two-step reaction, based on transcription with a bacteriophagepolymerase followed by translation in the rabbit reticulocyte lysate orwheat germ lysate. Because the transcription and translation reactionsare separate, each can be optimized to ensure that both are functioningat their full potential.

Unlike eukaryotic systems where transcription and translation occursequentially, in E. coli, transcription and translation occursimultaneously within the cell. In vitro E. coli translation systems arethus performed the same way, coupled, in the same tube under the samereaction conditions. During transcription, the 5′ end of the RNA becomesavailable for ribosomal binding and undergoes translation while its 3′end is still being transcribed. This early binding of ribosomes to theRNA maintains transcript stability and promotes efficient translation.This bacterial translation system gives efficient expression of eitherprokaryotic or eukaryotic gene products in a short amount of time. Forthe highest protein yield and the best initiation fidelity, one mayensure that the DNA template has a Shine-Dalgarno ribosome binding siteupstream of the initiator codon. Capping of eukaryotic RNA is notrequired. Use of E. coli extract also eliminates cross-reactivity orother problems associated with endogenous proteins in eukaryoticlysates. Also, the E. coli S30 extract system allows expression from DNAvectors containing natural E. coli promoter sequences (such as lac ortac). In various embodiments, the present methods employ a bactenophagepromoter (e.g., without limitation, T7, T3, or SP6). In variousembodiments, the present methods employ the TX-TL system as described inShin and Noireaux, J Biol. Eng. 4,8 (2010) and US Patent PL:blicationNo. 2016/0002611, the entire contents of which are hereby incorporatedby reference in their entireties.

In various embodiments, the nucleic acid encoding the candidateallosteric DNA-binding protein sensor and/or switch and a reporter genesystem comprises a single nucleic acid vector.

In various embodiments, the nucleic acid encoding the candidateallosteric DNA-binding protein sensor and/or switch and a reporter genesystem comprises two nucleic acid vectors. In an illustrativeembodiment, the protein sensor and/or switch, e.g. transcription factorlibrary, resides on a first plasmid while the reporter gene systemresides on a second plasmid. By having two separate plasmids, theeffective concentration of reporter gene to sensor library members maybe adjusted to facilitate identification of active library members. Thisis useful, for example where simply using higher versus lower promoterstrength is not enough control.

During the strain improvement process, it can be useful to rapidly swapfrom one sensor plasmid to another sensor plasmid. For instance, ahighly sensitive plasmid required for initial strain improvement maysaturate as the strain or strain library is improved. Rapidly swappingthe sensitive sensor plasmid for another harboring a less sensitiveplasmid facilitates further strain improvement. Another instance couldbe that the desired molecule to be sensed for further strain improvementmay change. To facilitate swapping between sensors, a sensor plasmid mayadditionally express a method directing the restriction of anothersensor plasmid. By having three or more unique targets it allow at willrestriction of any plasmid for another, i.e. Type A restriction targetsType B, Type B restriction targets Type C, and Type C targets Type A.

As used herein, a vector (or plasmid) refers to discrete elements thatare used to, for example, introduce heterologous nucleic acid into cellsfor expression or replication thereof. The vectors can remain episomalor can be designed to effect integration of a gene or portion thereofinto a chromosome of the genome. Also contemplated are vectors that areartificial chromosomes, such as yeast artificial chromosomes andmammalian artificial chromosomes. Selection and use of such vehicles arewell known to those of skill in the art. Included are vectors capable ofexpressing DNA that is operatively linked with regulatory sequences,such as promoter regions, that are capable of effecting expression ofsuch DNA fragments (e.g. expression vectors). Thus, a vector refers to arecombinant DNA or RNA construct, such as a plasmid, a phage,recombinant virus or other vector that, upon introduction into anappropriate host cell, results in expression of the DNA. Appropriatevectors are well known to those of skill in the art and include thosethat are replicable in eukaryotic cells and/or prokaryotic cells andthose that remain episomal or those that integrate into the host cellgenome.

In some embodiments, the present compositions and methods can includevectors based and/or generated using commercially available expressionconstructs, which can optionally be adapted or optimized for use incertain species and/or cell types. Examples of such expressionconstructs include the GATEWAY cloning vector available from INVITROGEN,which is available for multiple species. Examples of other expressionconstructs suitable for use in various species are known in the art. Byway of example, expression constructs suitable for use in, for example,Pichia pastoris include, for example, pAO815, pGAPZ, pGAPZa, pHIL-D2,pHIL-S1, pPIC3.5K, pPIC9K, pPICZ, and pPICZa. By way of example,expression constructs suitable for episomal maintenance in for example,Kluyveromyces lactis include, for example, pKD1. Expression constructssuitable for integration in Kluyveromyces lactis include, for example,pGB-HSb20 vector (Swinkels et al. Antonie van Leeuwenhoek, 64:187-201(1993); Bergkamp et al., Current Genetics, 21(4-5):365-370 (1992);Rossolini et al. Gene, 21; 119(1):75-81 (1992); Dominguez et al., theOfficial Journal of the Spanish Society for Microbiology, 1:131-142(1998)), pKLAC1 or pKLAC2 (Paul A. Colussi and Christopher H. Taron,Appl Environ Microbiol. 71(11): 7092-7098 (2005)).

The art provides a variety of vectors that find use in the presentinvention. By way of non-limiting illustration, phage vectors, plasmidvectors, phagemid vectors, phasmid vectors, cosmid vectors, virusvectors and YAC vectors may be used in the present invention.

Illustrative vectors are found in WO 2015/017866, e.g. at paragraphs[00154]-[00160], the entire contents of which are hereby incorporated byreference in its entirety.

Certain embodiments require the use of cloning methods, which are knownin the art and include, by way of non-limiting example, fusion PCR andassembly PCR see, e.g. Stemmer et al. Gene 164(1): 49-53 (1995), inversefusion PCR see, e.g. Spiliotis et al. PLoS ONE 7(4): 35407 (2012), sitedirected mutagenesis see, e.g. Ruvkun et al. Nature 289(5793): 85-88(1981), Gibson assembly (see, e.g. Gibson et al. Nature Methods 6 (5):343-345, (2009), the contents of which are hereby incorporated byreference in their entirety), Quick Change see, e.g. Kalnins et al. EMBO2(4): 593-7 (1983), Gateway see, e.g. Hartley et al. Genome Res.10(11):1788-95 (2000), Golden Gate see, e.g. Engler et al. Methods MolBiol. 1116:119-31 (2014), restriction digest and ligation including butnot Limited to blunt end, sticky end, and TA methods see, e.g. Cohen etal. PNAS 70 (11): 3240-4 (1973).

The invention is further described with reference to the followingnon-limiting examples.

EXAMPLES Example 1 Control of Transcription by an AllostericTranscription Factor In Vitro

To identify potential fluorogenic reporter molecule and enzyme pairs forcell-free aTF screening, the compounds fluoresceindi-(beta-D-glucopyranoside) (FDG) and fluorescein diphosphate (FDP) werescreen in vitro with their corresponding enzymes beta-glucosidase andAntarctic phosphatase. The following reactions were performed in 1× NEBPURE lysate to simulate the cell-free conditions in which they would bedeployed when selecting for engineered aTFs with novel ligand bindingactivity.

For FDG, reactions were initiated lx NEB PURE lysate spiked with 100 nMFDG substrate and a titrating amount of recombinant beta-glucosidaseenzyme from 1 mM to 0 mM in 2-fold dilutions. The reactions wereincubated at 37° C. for 8 hrs in a fluorescence plate reader, recordingthe fluorescence every 5 min. Decreasing the enzyme concentrationresulted in a linear decrease in the fluorescence production rate. Thisassay has a sensitivity to as low as 33 nM enzyme concentration with adynamic range greater than 30-fold (FIG. 6). There was no detectablebackground fluorescence production in the absence of exogenousbeta-glucosidase suggesting no enzymatic breakdown of the substrate bythe lysate constituents that could interfere with cell-free aTFscreening.

For FDP, reactions were initiated lx NEB PURE lysate spiked with 100 nMFDP substrate and a titrating amount of recombinant Antarcticphosphatase enzyme from 0.5 uU to 0 uU in 2-fold dilutions. Thereactions were incubated at 37° C. for 8 hours in a fluorescence platereader, recording the fluorescence every 5 min. Decreasing the enzymeconcentration resulted in a linear decrease in the fluorescenceproduction rate. This assay has a sensitivity to as low as 19 nM enzymeconcentration with a dynamic range greater than 75-fold (FIG. 7). Therewas no detectable background fluorescence production in the absence ofexogenous beta-glucosidase suggesting no enzymatic breakdown of thesubstrate by the lysate constituents that could interfere with cell-freeaTF screening.

The tetR gene was cloned into the pET28a(+) E. coli expression plasmidwith a C-terminal 6× his tag. The plasmid was cloned into BL21(DE3)cells inoculated into expression medium (LB+kanamycin) at a startingOD600 of 0.05 and grown to mid log phase. Cells were induced with 1 mMIPTG and protein expression occurred for 4 hours. After 4 hours, cellswere pelleted at 5, 000 rpm for 10 min, the supernatant was aspirated,and the cells were resuspended in TGN500 buffer [10 mM tris pH 7.5, 10%glycerol, 500 mM NaCl]. Cells were lysed by sonication following a 5 secon 55 sec off protocol for a total of 1 min on time. Cellular debris waspelleted by centrifugation at 12, 000 rpm for 30 min. Clarified lysatecontaining the recombinant tetR was incubated with nickel affinityresin, washed 10× with TGN500 buffer, and eluted stepwise withincreasing concentrations of imidazole. TetR eluted from the nickelcolumn with 150 mM imidazole in >95% purity.

The T7 RNA polymerase gene was cloned into the pET28a(+) E. coliexpression plasmid with a C-terminal 6× his tag. The plasmid was clonedinto BL21(DE3) cells inoculated into expression medium (LB+kanamycin) ata starting OD600 of 0.05 and grown to mid log phase. Cells were inducedwith 1 mM IPTG and protein expression occurred for 4 hours. After 4hours, cells were pelleted at 5, 000 rpm for 10 min, the supernatant wasaspirated, and the cells were resuspended in TGN500 buffer [10 mM trispH 7.5, 10% glycerol, 500 mM NaCI]. Cells were lysed by sonicationfollowing a 5 sec on 55 sec off protocol for a total of 1 min on time.Cellular debris was pelleted by centrifugation at 12, 000 rpm for 30min. Clarified lysate containing the recombinant tetR was incubated withnickel affinity resin, washed 10× with TGN500 buffer, and elutedstepwise with increasing concentrations of imidazole. T7 RNA polymeraseeluted from the nickel column with 150 mM imidazole in >95% purity.

A plasmid was constructed containing a T7 reporter construct [T7promoter upstream of a tetR operator followed by a tetR expressioncassette and a T7 terminator]. This reporter construct allows for tetRcontrolled of T7 amplification of the tetR gene. T7 transcription in 2×IVT mix [100 mM tris-HCI pH 7.5, 30 mM MgCl₂, 10 mM DTT, 4 mMspermidine, 5 mM each NTP, 4 U/uL RNase inhibitor, 4 U/uL T7 RNApolymerase] and diluted to lx with 100 nM final reporter plasmid asdescribed above, and a titration of purified tetR from 2 uM to 0 uM. Thereactions were incubated at 37° C. for 4 hours. Transcripts weredenatured in 2x RNA loading dye and run on a 1% agarose gel for 1 hr at90V constant, stained with SYBR Safe, and imaged on a gel doc. Effectivetranscription repression was seen with a stoichiometric amount of tetRas plasmid, in this case 100 nM plasmid and 100 nM tetR (FIG. 4, FIG.7). This data demonstrates the ability of tetR to repress T7 RNApolymerase activity in vitro.

Using a fixed amount of tetR and plasmid, 100 nM each, T7 transcriptionreactions were set up as described above with the inclusion of atitrating amount of anhydrotetracycline (ATC), the native ligand fortetR. The ATC titration ranged from 2 uM to 0 uM. The IVT transcriptswere analyzed by gel as described above. At low ATC concentrations,there was no generation of RNA transcripts suggesting completerepression of transcription by tetR in vitro. At a concentration of 2stoichiometric units of ATC (200 nM), a strong RNA band was generatedsimilar to that when no tetR in included in the reaction suggesting fulldepression of the tetR gene by the ligand in vitro (FIG. 9).Additionally, titrated amounts of ATC show a titratable amount of RNAproduced demonstrating the potential of a range of ligand bindingaffinities to produce differential amounts of RNA product (FIG. 4, FIG.7). This titratable response is a requirement when working withengineered sensors cell-free to enrich a population for those memberswith improved ligand binding function in the pool.

This strategy may be used in microfluidically generated emulsions asshown in (FIG. 4) or bulk emulsions and shown in (FIG. 2) for thescreening of engineered sensor activity in a cell-free context.

These results demonstrate the utility of aTFs in cell-free environmentsas well as the ability to screen for aTF activity using transcription asa response.

This is particularly useful in situations when, inter alia, a targetmolecule is toxic to cells in vivo and therefore a sensor to conductcell-based detection is impractical. By way of illustration, FIG. 10shows the dose response of 4 TetR sensors engineered to detect thetarget molecule nootkatone (CE3, GF1, GA3, and CG5) and wild type TetR(p523) to nootkatone and ATc. As seen in this cell-based assay, after0.5 mg/mL nootkatone, there is a toxic effect and the cells start todie. Accordingly, the detection of this target molecule would benefitfrom the cell-free methods described herein.

Example 2 Swapping a Primary Sensor Plasmid for a Secondary SensorPlasmid

As an example, a population of cells was generated with a primary sensorplasmid harboring a single I-Scel restriction enzyme cut site and anampicillin selection marker and expressing GFP (p1057). A secondarysensor plasmid was generated containing an expression cassette for theI-Scel enzyme and a kanamycin resistance cassette and RFP (p1174).Removal of the ampicillin from the selective medium did not result in astochastic removal of the primary sensor plasmid. Based on flowcytometry, no difference was observed between a clean background straintransformed only with p1174 and the strain harboring the p1057 plasmid.However, introduction of the secondary sensor plasmid and subsequentgrowth on kanamycin selective medium resulted in a 200, 000-foldreduction in cells harboring the primary plasmid in the population(FIGS. 11 and 12).

The following references are incorporated by reference in theirentireties:

J. R. Davis et al. Study of PcaV from Streptomyces coelicolor yields newinsights into ligand-responsive MarR family transcription factors. 2013,Nucleic Acids Research, 41(6) 3888-3900

S. Kosuri, et al. Composability of regulatory sequences controllingtranscription and translation in Escherichia coli. 2013, PNAS 110(34)14024-14029

D. L. Stauff and B. L. Bassler. Quorum Sensing in Chromobacteriumviolaceum: DNA Recognition and Gene Regulation by the CviR Receptor.2011 Journal of Bacteriology 193(15) 3871-3878

S. Grkovic, et al. The Staphylococcal QacR Multidrug Regulator Binds aCorrectly Spaced Operator as a Pair of Dimers. 2001 Journal ofBacteriology 183(24) 7102-7109

Z. Nie, et al. Polymer Particles with Various Shapes and MorphologiesProduced in Continuous Microfluidic Reactors. 2005, Journal of theAmerican Chemical Society 127 8058-63.

All of the numerical ranges, amounts, values and percentages, such asthose for amounts of materials, elemental contents, times andtemperatures of reaction, ratios of amounts, and others, in thefollowing portion of the specification and attached claims may be readas if prefaced by the word “about” even though the term “about” may notexpressly appear with the value, amount, or range. Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and attached claims are approximations that mayvary depending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains errornecessarily resulting from the standard deviation found in itsunderlying respective testing measurements. Furthermore, when numericalranges are set forth herein, these ranges are inclusive of the recitedrange end points (e.g., end points may be used). When percentages byweight are used herein, the numerical values reported are relative tothe total weight.

Also, it should be understood that any numerical range recited herein isintended to include all sub-ranges subsumed therein. For example, arange of “1 to 10” is intended to include all sub-ranges between (andincluding) the recited minimum value of 1 and the recited maximum valueof 10, that is, having a minimum value equal to or greater than 1 and amaximum value of equal to or less than 10. The terms “one, ” “a, ” or“an” as used herein are intended to include “at least one” or “one ormore, ” unless otherwise indicated.

Any aspect or embodiment disclosed herein can be combined with any otheraspect or embodiment as disclosed herein.

Any patent, publication, or other disclosure material, in whole or inpart, that is said to be incorporated by reference herein isincorporated herein only to the extent that the incorporated materialdoes not conflict with existing definitions, statements, or otherdisclosure material set forth in this disclosure. As such, and to theextent necessary, the disclosure as explicitly set forth hereinsupersedes any conflicting material incorporated herein by reference.Any material, or portion thereof, that is said to be incorporated byreference herein, but which conflicts with existing definitions,statements, or other disclosure material set forth herein will only beincorporated to the extent that no conflict arises between thatincorporated material and the existing disclosure material.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A method of making an allosteric DNA-bindingprotein sensor and/or switch which binds to a target molecule,comprising: (a) constructing a candidate allosteric DNA-binding proteinsensor and/or switch, the constructing comprising (i) designing aDNA-binding protein sensor and/or switch for an ability to bind a targetmolecule, the designing optionally being in silico or (ii) undertakingdirected or random mutagenesis to yield a candidate allostericDNA-binding protein sensor and/or switch having an ability to bind atarget molecule; (b) providing a host cell with a nucleic acid encodingthe candidate allosteric DNA-binding protein sensor and/or switch and anucleic acid encoding a reporter gene system and selecting for a cellcomprising the candidate allosteric DNA-binding protein sensor and/orswitch and the reporter gene system; (c) isolating nucleic acids fromthe cell comprising the candidate allosteric DNA-binding protein sensorand/or switch and the reporter gene system and contacting the isolatednucleic acids with an in vitro transcription (IVT) or an in vitrotranscription and translation (IVTT) mixture, the IVT or IVTT mixturecomprising a target molecule and a detection reagent; and (d)interrogating the IVT or IVTT mixture for reporter response, thereporter response being indicative of target molecule binding to thecandidate allosteric DNA-binding protein sensor and/or switch.
 2. Themethod of claim 1, wherein the allosteric DNA-binding protein sensorand/or switch is an engineered prokaryotic transcriptional regulatorfamily member optionally selected from a LysR, AraC/XylS, TetR, LuxR,Lacl, ArsR, MerR, AsnC, MarR, NtrC (EBP), OmpR, DeoR, Cold shock, GntR,and Crp family member.
 3. The method of claim 1 or 2, wherein the targetmolecule is a small molecule that is not a native ligand of the wildtype candidate allosteric DNA-binding protein sensor and/or switch. 4.The method of any one of the above claims, wherein the target moleculeis an antibiotic.
 5. The method of any one of the above claims, whereinstep (a) comprises mutating an allosteric protein.
 6. The method of anyone of the above claims, wherein the nucleic acid is provided to thehost cell by one or more of electroporation, chemical transformation,ballistic transformation, pressure induced transformation, electrosprayinjection, mechanical shear forces induced, for example, in microfluids,and carbon nanotubes, nanotube puncture, induced natural competencemechanisms of an organism, merging of protoplasts, and conjugation withAgrobacterium.
 7. The method of any one of the above claims, wherein thehost cell is selected from a eukaryotic or prokaryotic cell, selectedfrom a bacterial, yeast, algal, plant, insect, mammalian cells, andimmortalized cell.
 8. The method of any one of the above claims, whereinthe reporter gene system comprises a protein having a unique spectralsignature and/or assayable enzymatic activity.
 9. The method of any oneof the above claims, wherein the IVT or IVTT mixture comprises a coupledor linked system.
 10. The method of any one of the above claims, whereinthe reporterresponse is a direct amplification of the genotype of theallosteric protein.
 11. The method of any one of the above claims,wherein the nucleic acid encoding the candidate allosteric DNA-bindingprotein sensor and/or switch and the nucleic acid encoding the reportergene system comprises a single nucleic acid vector.
 12. The method ofany one of the above claims, wherein the nucleic acid encoding thecandidate allosteric DNA-binding protein sensor and/or switch and thenucleic acid encoding the reporter gene system comprises two nucleicacid vectors.
 13. The method of any one of the above claims, furthercomprising: (e) isolating the nucleic acid encoding the candidateallosteric DNA-binding protein sensor and/or switch.
 14. The method ofclaim 13, wherein the isolating comprises the use of flasks, culturetubes, and plastic ware, microliter plates, patterned microwells, ormicrodroplets generated either in bulk or microfluidically.
 15. A methodof making an allosteric DNA-binding protein sensor and/or switch whichbinds to a target molecule, comprising: (a) constructing a candidateallosteric DNA-binding protein sensor and/or switch, the constructingcomprising (i) designing a DNA-binding protein sensor and/or switch foran ability to bind a target molecule, the designing optionally being insilico or (ii) undertaking directed or random mutagenesis to yield aDNA-binding protein sensor and/or switch which has an ability to bind atarget molecule; (b) providing a host cell with a nucleic acid encodingthe candidate allosteric DNA-binding protein sensor and/or switch and anucleic acid encoding a reporter gene system and selecting for a cellcomprising the candidate allosteric DNA-binding protein sensor and/orswitch and the reporter gene system; (c) isolating nucleic acids fromthe cell comprising the candidate allosteric DNA-binding protein sensorand/or switch and the reporter gene system and contacting the isolatednucleic acids with an in vitro transcription (IVT) or an in vitrotranscription and translation (IVTT) mixture, the IVT or IVTT mixturecomprising a target molecule and a detection reagent; and (d)interrogating the IVT or IVTT mixture by nucleic acid sequencing beforeand after selection to determine those molecules that have becomefunctionally enriched.
 16. The method of claim 15, wherein theallosteric DNA-binding protein sensor and/or switch is an engineeredprokaryotic transcriptional regulator family member optionally selectedfrom a LysR, AraC/XylS, TetR, LuxR, Lacl, ArsR, MerR, AsnC, MarR, NtrC(EBP), OmpR, DeoR, Cold shock, GntR, and Crp family member.
 17. Themethod of claim 15 or 16, wherein the target molecule is a smallmolecule that is not a native ligand of the wild type candidateallosteric DNA-binding protein sensor and/or switch.
 18. The method ofany one of claims 15-17, wherein the target molecule is an antibiotic.19. The method of any one of claims 15-18, wherein step (a) comprisesmutating an allosteric protein.
 20. The method of any one of claims15-19, wherein the nucleic acid is provided to the host cell by one ormore of electroporation, chemical transformation, ballistictransformation, pressure induced transformation, electrospray injection,mechanical shear forces induced, for example, in microfluids, and carbonnanotubes, nanotube puncture, induced natural competence mechanisms ofan organism, merging of protoplasts, and conjugation with Agrobacterium.21. The method of any one of claims 15-20, wherein the host cell isselected from a eukaryotic or prokaryotic cell, selected from abacterial, yeast, algal, plant, insect, mammalian cells, andimmortalized cell.
 22. The method of any one of claims 15-21, whereinthe reporter gene system comprises a protein having a unique spectralsignature and/or assayable enzymatic activity.
 23. The method of any oneof claims 15-22, wherein the IVT or IVTT mixture comprises a coupled orlinked system.
 24. The method of any one of claims 15-23, wherein thereporter response is a direct amplification of the genotype of theallosteric protein.
 25. The method of any one of claims 15-24, whereinthe nucleic acid encoding the candidate allosteric DNA-binding proteinsensor and/or switch and the nucleic acid encoding the reporter genesystem comprises a single nucleic acid vector.
 26. The method of any oneof claims 15-25, wherein the nucleic acid encoding the candidateallosteric DNA-binding protein sensor and/or switch and the nucleic acidencoding the reporter gene system comprises two nucleic acid vectors.27. The method of any one of claims 15-26, further comprising: (e)isolating the nucleic acid encoding the candidate allosteric DNA-bindingprotein sensor and/or switch.
 28. The method of claim 27, wherein theisolating comprises the use of flasks, culture tubes, and plastic ware,microliter plates, patterned microwells, or microdroplets generatedeither in bulk or microfluidically.
 29. A method of making an allostericDNA-binding protein sensor and/or switch which binds to a targetmolecule, comprising: (a) constructing a candidate allostericDNA-binding protein sensor and/or switch, the constructing comprising(i) designing a DNA-binding protein sensor and/or switch for an abilityto bind a target molecule, the designing optionally being in silico or(ii) undertaking directed or random mutagenesis to yield the candidateallosteric DNA-binding protein sensor and/or switch having an ability tobind a target molecule; (b) contacting a solid support with a nucleicacid encoding the candidate allosteric DNA-binding protein sensor and/orswitch and selecting for a solid support comprising the candidateallosteric DNA-binding protein sensor and/or switch; (c) isolatingnucleic acids from the solid support comprising the candidate allostericDNA-binding protein sensor and/or switch and contacting the isolatednucleic acids with an in vitro transcription (IVT) or an in vitrotranscription and translation (IVTT) mixture; (d) introducing a reportergene system, detection reagent, and target molecule, and interrogatingthe mixture for a reporter response, the reporter response beingindicative of the target molecule binding to the candidate allostericDNA-binding protein sensor and/or switch.
 30. The method of claim 29,wherein the solid support is a nanoparticle and a microparticle.
 31. Themethod of claim 29, wherein the solid support is a bead, selected from ananobead and a microbead.
 32. The method of claim 29, wherein the solidsupport is an array.
 33. The method of any one of claims 29-32, whereinthe candidate allosteric DNA-binding protein sensor and/or switch is anengineered prokaryotic transcriptional regulator family memberoptionally selected from a LysR, AraC/XylS, TetR, LuxR, Lacl, ArsR,MerR, AsnC, MarR, NtrC (EBP), OmpR, DeoR, Cold shock, GntR, and Crpfamily member.
 34. The method of any one of claims 29-33, wherein thetarget molecule is a small molecule that is not a native ligand of thewild type candidate allosteric DNA-binding protein sensor and/or switch.35. The method of any one of claims 29-33, wherein the target moleculeis an antibiotic.
 36. The method of any one of claims 29-35, whereinstep (a) comprises mutating an allosteric protein.
 37. The method of anyone of claims 29-36, wherein the reporter gene system comprises aprotein having a unique spectral signature and/or assayable enzymaticactivity.
 38. The method of any one of claims 29-37, wherein the IVT orIVTT mixture comprises a coupled or linked system.
 39. The method of anyone of claims 29-38, wherein the reporter response is a directamplification of the genotype of the allosteric protein.
 40. The methodof any one of claims 29-39, wherein the nucleic acid encoding thecandidate allosteric DNA-binding protein sensor and/or switch and thenucleic acid encoding the reporter gene system comprises a singlenucleic acid vector.
 41. The method of any one of claims 29-39, whereinthe nucleic acid encoding the candidate allosteric DNA-binding proteinsensor and/or switch and the nucleic acid encoding the reporter genesystem comprises two nucleic acid vectors.
 42. The method of any one ofclaims 29-41, wherein the nucleic acid encoding the candidate allostericDNA-binding protein sensor and/or switch comprises a synthetic DNA,amplified DNA, or amplified RNA.
 43. The method of any one of claims29-42, further comprising: (e) isolating the nucleic acid encoding theallosteric DNA-binding protein sensor and/or switch.
 44. The method ofclaim 43, wherein the isolating comprises the use of flasks, culturetubes, and plastic ware, microliter plates, patterned microwells, ormicrodroplets generated either in bulk or microfluidically.
 45. A methodfor making a target molecule in a biological cell, comprising: (a)engineering the biological cell to produce the target molecule; (b)introducing an allosteric DNA-binding protein sensor and/or switch whichbinds to the target molecule in the biological cell; and (c) screeningfor target molecule production.
 46. The method of claim 45, wherein thebiological cell is engineered to produce the target molecule by amultiplex genome engineering technique and/or a method involving adouble-strand break (DSB) or single-strand break or nick.
 47. The methodof claim 45 or 46, wherein the allosteric DNA-binding protein sensorand/or switch which binds to the target molecule is produced by a methodcomprising: (a) constructing a candidate allosteric DNA-binding proteinsensor and/or switch, the constructing comprising (i) designing acandidate allosteric DNA-binding protein sensor and/or switch for anability to bind the target molecule, the designing optionally being insilico or (ii) undertaking directed or random mutagenesis to yield thecandidate allosteric DNA-binding protein sensor and/or switch having anability to bind the target molecule; (b) providing a host cell with anucleic acid encoding the candidate allosteric DNA-binding proteinsensor and/or switch and a nucleic acid encoding the reporter genesystem and selecting for a cell comprising the candidate allostericDNA-binding protein sensor and/or switch and the reporter gene system;(c) isolating nucleic acids from the cell comprising the candidateallosteric DNA-binding protein sensor and/or switch and the reportergene system and contacting the isolated nucleic acids with an in vitrotranscription (IVT) or an in vitro transcription and translation (IVTT)mixture, the IVT or IVTT mixture comprising a target molecule and adetection reagent; and (d) interrogating the IVT or IVTT mixture forreporter response, the reporter response being indicative of targetmolecule binding to the allosteric DNA-binding protein sensor and/orswitch.
 48. The method of claim 45 or 46, wherein the allostericDNA-binding protein sensor and/or switch which binds to the targetmolecule is produced by a method comprising: (a) constructing acandidate allosteric DNA-binding protein sensor and/or switch, theconstructing comprising (i) designing a candidate allosteric DNA-bindingprotein sensor and/or switch for an ability to bind the target molecule,the designing optionally being in silico or (ii) undertaking directed orrandom mutagenesis to yield the candidate allosteric DNA-binding proteinsensor and/or switch which has an ability to bind the target molecule;(b) providing a host cell with a nucleic acid encoding the candidateallosteric DNA-binding protein sensor and/or switch and the reportergene system and selecting for a cell comprising the candidate allostericDNA-binding protein sensor and/or switch and the reporter gene system;(c) isolating nucleic acids from the cell comprising the candidateallosteric DNA-binding protein sensor and/or switch and the reportergene system and contacting the isolated nucleic acids with an in vitrotranscription (IVT) or an in vitro transcription and translation (IVTT)mixture, the IVT or IVTT mixture comprising a target molecule and adetection reagent; and (d) interrogating the IVT or IVTT mixture bynucleic acid sequencing before and after selection to determine thosemolecules that have become functionally enriched.
 49. The method ofclaim 45 or 46, wherein the allosteric DNA-binding protein sensor and/orswitch which binds to a target molecule is produced by a methodcomprising: (a) constructing a candidate allosteric DNA-binding proteinsensor and/or switch, the constructing comprising (i) designing acandidate allosteric DNA-binding protein sensor and/or switch for anability to bind the target molecule, the designing optionally being insilico or (ii) undertaking directed or random mutagenesis to yield thecandidate allosteric DNA-binding protein sensor and/or switch having anability to bind the target molecule; (b) contacting a solid support witha nucleic acid encoding the candidate allosteric DNA-binding proteinsensor and/or switch and selecting for a solid support comprising thecandidate allosteric DNA-binding protein sensor and/or switch; (c)isolating nucleic acids from the solid support comprising the candidateallosteric DNA-binding protein sensor and/or switch and contacting theisolated nucleic acids with an in vitro transcription (IVT) or an invitro transcription and translation (IVTT) mixture; (d) introducing areporter gene system, detection reagent, and target molecule, andinterrogating the mixture for a reporter response, the reporter responsebeing indicative of target molecule binding to the candidate allostericDNA-binding protein sensor and/or switch.
 50. The method of claim 49,wherein the solid support is a nanoparticle and a microparticle.
 51. Themethod of claim 49, wherein the solid support is a bead, selected from ananobead and a microbead.
 52. The method of claim 49, wherein the solidsupport is an array.
 53. The method of any one of claims 45-52, whereinthe allosteric DNA-binding protein sensor and/or switch is an engineeredprokaryotic transcriptional regulator family member optionally selectedfrom a LysR, AraC/XylS, TetR, LuxR, Lacl, ArsR, MerR, AsnC, MarR, NtrC(EBP), OmpR, DeoR, Cold shock, GntR, and Crp family member.
 55. Themethod of any one of claims 45-53, wherein the screening for targetmolecule comprises a positive or negative screen.
 56. The method of anyone of claims 45-55, wherein the allosteric DNA-binding protein sensorand/or switch is one or more of those of Table 1 and has about 1, or 2,or 3, or 4, or 5, or 10 mutations.